2-azabicyclo hexane jak inhibitor compound

ABSTRACT

The invention provides a compound of formula 1 
     
       
         
         
             
             
         
       
     
     or a pharmaceutically-acceptable salt thereof, that is useful as a JAK inhibitor. The invention also provides pharmaceutical compositions comprising the compound, methods of using the compound to treat diseases amenable to a JAK inhibitor, and processes useful for preparing the compound.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims the benefit of U.S. Provisional Application No.62/751,967, filed on Oct. 29, 2018, the disclosure of which isincorporated herein by reference in its entirety.

BACKGROUND OF THE INVENTION Field of the Invention

The invention is directed to a JAK kinase inhibitor compound useful forthe treatment of inflammatory diseases, particularly ocular diseases.The invention is also directed to pharmaceutical compositions comprisingsuch a compound, methods of using such a compound to treat oculardiseases, and processes useful for preparing the compound.

State of the Art

Cytokines are intercellular signaling molecules which includechemokines, interferons, interleukins, lymphokines, and tumour necrosisfactor. Cytokines are critical for normal cell growth andimmunoregulation but also drive immune-mediated diseases and contributeto the growth of malignant cells. Elevated levels of many cytokines havebeen implicated in the pathology of a large number of diseases orconditions, particularly those diseases characterized by inflammation.Many of the cytokines implicated in disease act through signalingpathways dependent upon the Janus family of tyrosine kinases (JAKs),which signal through the Signal Transducer and Activator ofTranscription (STAT) family of transcription factors.

The JAK family comprises four members, JAK1, JAK2, JAK3, and tyrosinekinase 2 (TYK2). Binding of cytokine to a JAK-dependent cytokinereceptor induces receptor dimerization which results in phosphorylationof tyrosine residues on the JAK kinase, effecting JAK activation.Phosphorylated JAKs, in turn, bind and phosphorylate various STATproteins which dimerize, internalize in the cell nucleus and directlymodulate gene transcription, leading, among other effects, to thedownstream effects associated with inflammatory disease. The JAKsusually associate with cytokine receptors in pairs as homodimers orheterodimers. Specific cytokines are associated with specific JAKpairings. Each of the four members of the JAK family is implicated inthe signaling of at least one of the cytokines associated withinflammation.

Inflammation plays a prominent role in many ocular diseases, includinguveitis, diabetic retinopathy, diabetic macular edema, dry eye disease,age-related macular degeneration, retinal vein occlusion and atopickeratoconjunctivitis. Uveitis encompasses multiple intraocularinflammatory conditions and is often autoimmune, arising without a knowninfectious trigger. The condition is estimated to affect about 2 millionpatients in the US. In some patients, the chronic inflammationassociated with uveitis leads to tissue destruction, and it is the fifthleading cause of blindness in the US. Cytokines elevated in uveitispatients' eyes that signal through the JAK-STAT pathway include IL-2,IL-4, IL-5, IL-6, IL-10, IL-23, and IFN-γ. (Horai and Caspi, JInterferon Cytokine Res, 2011, 31, 733-744; Ooi et al, Clinical Medicineand Research, 2006, 4, 294-309). Existing therapies for uveitis areoften suboptimal, and many patients are poorly controlled. Steroids,while often effective, are associated with cataracts and increasedintraocular pressure/glaucoma.

Diabetic retinopathy (DR) is caused by damage to the blood vessels inthe retina. It is the most common cause of vision loss among people withdiabetes. Angiogenic as well as inflammatory pathways play an importantrole in the disease. Often, DR will progress to diabetic macular edema(DME), the most frequent cause of visual loss in patients with diabetes.The condition is estimated to affect about 1.5 million patients in theUS alone, of whom about 20% have disease affecting both eyes. Cytokineswhich signal through the JAK-STAT pathway, such as IL-6, as well asother cytokines, such as IP-10 and MCP-1 (alternatively termed CCL2),whose production is driven in part by JAK-STAT pathway signaling, arebelieved to play a role in the inflammation associated with DR/DME(Abcouwer, J Clin Cell Immunol, 2013, Suppl 1, 1-12; Sohn et al.,American Journal of Opthalmology, 2011, 152, 686-694; Owen and Hartnett,Curr Diab Rep, 2013, 13, 476-480; Cheung et al, Molecular Vision, 2012,18, 830-837; Dong et al, Molecular Vision, 2013, 19, 1734-1746; Funatsuet al, Ophthalmology, 2009, 116, 73-79). The existing therapies for DMEare suboptimal: intravitreal anti-VEGF treatments are only effective ina fraction of patients and steroids are associated with cataracts andincreased intraocular pressure.

Dry eye disease (DED) is a multifactorial disorder that affectsapproximately 5 million patients in the US. Ocular surface inflammationis believed to play an important role in the development and propagationof this disease. Elevated levels of cytokines such as IL-1, IL-2, IL-4,IL-5, IL-6, and IFN-γ have been noted in the ocular fluids of patientswith DED. (Stevenson et al, Arch Ophthalmol, 2012, 130, 90-100), and thelevels often correlated with disease severity. Age-related maculardegeneration and atopic keratoconjunctivitis are also thought to beassociated with JAK-dependent cytokines.

Retinal vein occlusion (RVO) is a highly prevalent visually disablingdisease. Obstruction of retinal blood flow can lead to damage of theretinal vasculature, hemorrhage, and tissue ischemia. Although thecauses for RVO are multifactorial, both vascular as well as inflammatorymediators have been shown to be important (Deobhakta et al,International Journal of Inflammation, 2013, article ID 438412).Cytokines which signal through the JAK-STAT pathway, such as IL-6 andIL-13, as well as other cytokines, such as MCP-1, whose production isdriven in part by JAK-STAT pathway signaling, have been detected atelevated levels in ocular tissues of patients with RVO (Shchuko et al,Indian Journal of Ophthalmology, 2015, 63(12), 905-911). While manypatients with RVO are treated by photocoagulation, this is an inherentlydestructive therapy. Anti-VEGF agents are also used, but they are onlyeffective in a fraction of patients. Steroid medications that reduce thelevel of inflammation in the eye (Triamcinolone acetonide anddexamethasone implants) have also been shown to provide beneficialresults for patients with certain forms of RVO, but they have also beenshown to cause cataracts and increased intraocular pressure/glaucoma.

The need remains for a potent pan-JAK inhibitor for the treatment ofocular diseases.

SUMMARY OF THE INVENTION

In one aspect, the invention provides a JAK inhibitor compound usefulfor the treatment of ocular inflammatory disease.

In particular, in one aspect, the invention provides a compound of theformula:

hereinafter compound 1, or a pharmaceutically-acceptable salt thereof.

The invention also provides a pharmaceutical composition comprisingcompound 1, or a pharmaceutically acceptable salt thereof, and apharmaceutically-acceptable carrier.

In one aspect, the invention provides a method of treating an oculardisease in a mammal, the method comprising administering to the mammalcompound 1, or a pharmaceutical composition of the invention. In oneaspect the ocular disease is selected from the group consisting ofuveitis, diabetic retinopathy, diabetic macular edema, dry eye disease,age-related macular degeneration, retinal vein occlusion and atopickeratoconjunctivitis. In particular, the ocular disease is diabeticmacular edema or uveitis.

In separate and distinct aspects, the invention also provides syntheticprocesses described herein, which are useful for preparing compound 1.

The invention also provides compound 1, or a pharmaceutically acceptablesalt thereof, as described herein for use in medical therapy, as well asthe use of the compound of the invention in the manufacture of aformulation or medicament for treating ocular diseases.

DETAILED DESCRIPTION OF THE INVENTION

Chemical structures are named herein according to IUPAC conventions asimplemented in ChemDraw software (PerkinElmer, Inc., Cambridge, Mass.).

Furthermore, the imidazo portion of the tetrahydroimidazopyridine moietyin the structure of the present compound exists in tautomeric forms. Thecompound could equivalently be represented as

According to the IUPAC convention, these representations give rise todifferent numbering of the atoms of the tetrahydroimidazopyridineportion. Accordingly this structure is designated((1S,5R)-2-azabicyclo[3.1.0]hexan-1-yl)(2-(6-(2-ethyl-5-fluoro-4-hydroxyphenyl)-4-fluoro-1H-indazol-3-yl)-3,4,6,7-tetrahydro-5H-imidazo[4,5-c]pyridin-5-yl)methanone.It can also be designated:((1S,5R)-2-azabicyclo[3.1.0]hexan-1-yl)(2-(6-(2-ethyl-5-fluoro-4-hydroxyphenyl)-4-fluoro-1H-indazol-3-yl)-1,4,6,7-tetrahydro-5H-imidazo[4,5-c]pyridin-5-yl)methanone.It will be understood that although structures are shown, or named, in aparticular form, the invention also includes the tautomer thereof.

The compounds of the invention contain several basic groups andtherefore, the compounds can exist as the free base or in various saltforms, such as a mono-protonated salt form, a di-protonated salt form,or mixtures thereof. All such forms are included within the scope ofthis invention, unless otherwise indicated.

This invention also includes isotopically-labeled compounds of formula1, i.e., compounds of formula 1 where an atom has been replaced orenriched with an atom having the same atomic number but an atomic massdifferent from the atomic mass that predominates in nature. Examples ofisotopes that may be incorporated into a compound of formula 1 include,but are not limited to, ²H, ³H, ¹¹C, ¹³C, ¹⁴C, ¹³N, ¹⁵N, ¹⁵O, ¹⁷O, ¹⁸O,and ¹⁸F. Of particular interest are compounds of formula 1 enriched intritium or carbon-14, which compounds can be used, for example, intissue distribution studies. Also of particular interest are compoundsof formula 1 enriched in deuterium especially at a site of metabolism,which compounds are expected to have greater metabolic stability.Additionally of particular interest are compounds of formula 1 enrichedin a positron emitting isotope, such as ¹¹C, ¹⁸F, ¹⁵O and ¹³N, whichcompounds can be used, for example, in Positron Emission Tomography(PET) studies.

Definitions

When describing this invention including its various aspects andembodiments, the follow

The term “therapeutically effective amount” means an amount sufficientto effect treatment when administered to a patient in need of treatment.

The term “treating” or “treatment” means preventing, ameliorating orsuppressing the medical condition, disease or disorder being treated(e.g., a respiratory disease) in a patient (particularly a human); oralleviating the symptoms of the medical condition, disease or disorder.

The term “pharmaceutically acceptable salt” means a salt that isacceptable for administration to a patient or a mammal, such as a human(e.g., salts having acceptable mammalian safety for a given dosageregime). Representative pharmaceutically acceptable salts include saltsof acetic, ascorbic, benzenesulfonic, benzoic, camphorsulfonic, citric,ethanesulfonic, edisylic, fumaric, gentisic, gluconic, glucoronic,glutamic, hippuric, hydrobromic, hydrochloric, isethionic, lactic,lactobionic, maleic, malic, mandelic, methanesulfonic, mucic,naphthalenesulfonic, naphthalene-1,5-disulfonic,naphthalene-2,6-disulfonic, nicotinic, nitric, orotic, pamoic,pantothenic, phosphoric, succinic, sulfuric, tartaric, p-toluenesulfonicand xinafoic acid, and the like.

The term “salt thereof” means a compound formed when the hydrogen of anacid is replaced by a cation, such as a metal cation or an organiccation and the like. For example, the cation can be a protonated form ofa compound of formula 1, i.e. a form where one or more amino groups havebeen protonated by an acid. Typically, the salt is a pharmaceuticallyacceptable salt, although this is not required for salts of intermediatecompounds that are not intended for administration to a patient.

The term “amino-protecting group” means a protecting group suitable forpreventing undesired reactions at an amino nitrogen. Representativeamino-protecting groups include, but are not limited to, formyl; acylgroups, for example alkanoyl groups, such as acetyl andtri-fluoroacetyl; alkoxycarbonyl groups, such as tert butoxycarbonyl(Boc); arylmethoxycarbonyl groups, such as benzyloxycarbonyl (Cbz) and9-fluorenylmethoxycarbonyl (Fmoc); arylmethyl groups, such as benzyl(Bn), trityl (Tr), and 1,1-di-(4′-methoxyphenyl)methyl; silyl groups,such as trimethylsilyl (TMS), tert-butyldimethylsilyl (TBDMS),[2-(trimethylsilyl)ethoxy]methyl (SEM); and the like.

Numerous protecting groups, and their introduction and removal, aredescribed in T. W. Greene and P. G. M. Wuts, Protecting Groups inOrganic Synthesis, Third Edition, Wiley, New York

General Synthetic Procedures

Compound 1, and intermediates thereof, can be prepared according to thefollowing general methods and procedures using commercially-available orroutinely-prepared starting materials and reagents. Additionally,compounds having an acidic or basic atom or functional group may be usedor may be produced as a salt unless otherwise indicated (in some cases,the use of a salt in a particular reaction will require conversion ofthe salt to a non-salt form, e.g., a free base, using routine proceduresbefore conducting the reaction).

Although a particular embodiment of the present invention may be shownor described in the following procedures, those skilled in the art willrecognize that other embodiments or aspects of the present invention canalso be prepared using such procedures or by using other methods,reagents, and starting materials know to those skilled in the art. Inparticular, it will be appreciated that compound 1 may be prepared by avariety of process routes in which reactants are combined in differentorders to provide different intermediates en route to producing finalproducts.

The preparation of compound 1 is described in detail in the appendedexamples. Key steps are summarized in Scheme 1. R^(A) may be hydroxyl inwhich case, compound 7-PG is coupled with intermediate 6 under typicalamide bond formation conditions in the presence of an activating reagentsuch as HATU, HOBT and the like. Alternatively, R^(A) may be a leavinggroup such as Cl.

After coupling compound 6 with 7-PG, the protecting group “PG” isremoved to give compound 1. The protecting group can be selected fromamino-protecting groups as defined above. For example PG can be aBoc-protecting group, in which case the deprotection can be conducted inthe presence of a strong acid such as TFA or HCl.

Intermediate 3 may be prepared as described in the experimental section.An alternative method of preparation of the key protected intermediate 5is illustrated in Scheme 2.

The bromoindazole aldehyde 8 may be reacted with the benzyl protectedimine compound 2 to provide intermediate 9. The reaction is typicallyconducted in the presence of sodium bisulfite, at a temperature ofbetween about 130° C. and about 140° C. for between about 1 and about 6hours or until the reaction is substantially complete. Compound 9 isreduced using a reducing agent such as sodium borohydride to providecompound 10, which is combined with protected phenyltrifluoroborate 11under typical Suzuki-Miyaura coupling conditions to provide intermediate5. The reaction is typically conducted at elevated temperature in thepresence of a palladium catalyst. The Suzuki partner 11, shown in Scheme2 as the trifluoroborate potassium salt can be prepared by reacting thecorresponding boronate (Intermediate 1-5 in Preparation 1 below) withpotassium hydrogen difluoride to provide intermediate 11. Alternatively,the boronate intermediate can be used in place of the trifluoroborate11.

Accordingly, in a method aspect, the invention provides a process ofpreparing a compound of formula 1, or a pharmaceutically acceptable saltthereof, the process comprising reacting a compound of formula 6 with acompound of formula 7-PG, followed by removal of the protecting groupPG, and optionally preparing a pharmaceutically-acceptable salt ofcompound 1 to provide a compound of formula 1, or a pharmaceuticallyacceptable salt thereof.

Pharmaceutical Compositions

Compound 1, and pharmaceutically-acceptable salts thereof are typicallyused in the form of a pharmaceutical composition or formulation. Suchpharmaceutical compositions may advantageously be administered to apatient by any acceptable route of administration including, but notlimited to, oral, inhalation, optical injection, topical (includingtransdermal), rectal, nasal, and parenteral modes of administration.

Accordingly, in one of its compositions aspects, the invention isdirected to a pharmaceutical composition comprising apharmaceutically-acceptable carrier or excipient and compound 1, where,as defined above, “compound 1” means compound 1 or apharmaceutically-acceptable salt thereof. Optionally, suchpharmaceutical compositions may contain other therapeutic and/orformulating agents if desired. When discussing compositions and usesthereof, compound 1 may also be referred to herein as the “activeagent”.

In some aspects, the disclosure provides a pharmaceutical compositioncomprising compound 1, or a pharmaceutically acceptable salt thereof,and a pharmaceutically-acceptable carrier. In some aspects, thepharmaceutical composition is suitable for application to the eye. Insome aspects, the composition is suitable for injection into the eye. Insome aspects, the composition is suitable for intravitreal injection. Insome aspects, the composition is a suspension. In some aspects, thecomposition is a crystalline suspension.

The pharmaceutical compositions of the invention typically contain atherapeutically effective amount of compound 1. Those skilled in the artwill recognize, however, that a pharmaceutical composition may containmore than a therapeutically effective amount, i.e., bulk compositions,or less than a therapeutically effective amount, i.e., individual unitdoses designed for multiple administration to achieve a therapeuticallyeffective amount.

Typically, such pharmaceutical compositions will contain from about 0.01to about 95% by weight of the active agent; including, for example, fromabout 0.05 to about 30% by weight; and from about 0.1% to about 10% byweight of the active agent.

Any conventional carrier or excipient may be used in the pharmaceuticalcompositions of the invention. The choice of a particular carrier orexcipient, or combinations of carriers or excipients, will depend on themode of administration being used to treat a particular patient or typeof medical condition or disease state. In this regard, the preparationof a suitable pharmaceutical composition for a particular mode ofadministration is well within the scope of those skilled in thepharmaceutical arts. Additionally, the carriers or excipients used inthe pharmaceutical compositions of this invention arecommercially-available. By way of further illustration, conventionalformulation techniques are described in Remington: The Science andPractice of Pharmacy, 20th Edition, Lippincott Williams & White,Baltimore, Md. (2000); and H. C. Ansel et al., Pharmaceutical DosageForms and Drug Delivery Systems, 7th Edition, Lippincott Williams &White, Baltimore, Md. (1999).

Representative examples of materials which can serve as pharmaceuticallyacceptable carriers include, but are not limited to, the following:sugars, such as lactose, glucose and sucrose; starches, such as cornstarch and potato starch; cellulose, such as microcrystalline cellulose,and its derivatives, such as sodium carboxymethyl cellulose, ethylcellulose and cellulose acetate; powdered tragacanth; malt; gelatin;talc; excipients, such as cocoa butter and suppository waxes; oils, suchas peanut oil, cottonseed oil, safflower oil, sesame oil, olive oil,corn oil and soybean oil; glycols, such as propylene glycol; polyols,such as glycerin, sorbitol, mannitol and polyethylene glycol; esters,such as ethyl oleate and ethyl laurate; agar; buffering agents, such asmagnesium hydroxide and aluminum hydroxide; alginic acid; pyrogen-freewater; isotonic saline; Ringer's solution; ethyl alcohol; phosphatebuffer solutions; and other non-toxic compatible substances employed inpharmaceutical compositions.

Pharmaceutical compositions are typically prepared by thoroughly andintimately mixing or blending the active agent with apharmaceutically-acceptable carrier and one or more optionalingredients. The resulting uniformly blended mixture can then be shapedor loaded into tablets, capsules, pills and the like using conventionalprocedures and equipment.

The pharmaceutical compositions of the invention are preferably packagedin a unit dosage form. The term “unit dosage form” refers to aphysically discrete unit suitable for dosing a patient, i.e., each unitcontaining a predetermined quantity of active agent calculated toproduce the desired therapeutic effect either alone or in combinationwith one or more additional units. For example, such unit dosage formsmay be capsules, tablets, pills, and the like, or unit packages suitablefor ocular or parenteral administration.

In one embodiment, the pharmaceutical compositions of the invention aresuitable for oral administration. Suitable pharmaceutical compositionsfor oral administration may be in the form of capsules, tablets, pills,lozenges, cachets, dragees, powders, granules; or as a solution or asuspension in an aqueous or non-aqueous liquid; or as an oil-in-water orwater-in-oil liquid emulsion; or as an elixir or syrup; and the like;each containing a predetermined amount of compound 1 as an activeingredient.

When intended for oral administration in a solid dosage form (i.e., ascapsules, tablets, pills and the like), the pharmaceutical compositionsof the invention will typically comprise the active agent and one ormore pharmaceutically-acceptable carriers. Optionally, such solid dosageforms may comprise: fillers or extenders, such as starches,microcrystalline cellulose, lactose, dicalcium phosphate, sucrose,glucose, mannitol, and/or silicic acid; binders, such ascarboxymethylcellulose, alginates, gelatin, polyvinyl pyrrolidone,sucrose and/or acacia; humectants, such as glycerol; disintegratingagents, such as crosscarmellose sodium, agar-agar, calcium carbonate,potato or tapioca starch, alginic acid, certain silicates, and/or sodiumcarbonate; solution retarding agents, such as paraffin; absorptionaccelerators, such as quaternary ammonium compounds; wetting agents,such as cetyl alcohol and/or glycerol monostearate; absorbents, such askaolin and/or bentonite clay; lubricants, such as talc, calciumstearate, magnesium stearate, solid polyethylene glycols, sodium laurylsulfate, and/or mixtures thereof; coloring agents; and buffering agents.

Release agents, wetting agents, coating agents, sweetening, flavoringand perfuming agents, preservatives and antioxidants can also be presentin the pharmaceutical compositions of the invention. Examples ofpharmaceutically-acceptable antioxidants include: water-solubleantioxidants, such as ascorbic acid, cysteine hydrochloride, sodiumbisulfate, sodium metabisulfate, sodium sulfite and the like;oil-soluble antioxidants, such as ascorbyl palmitate, butylatedhydroxyanisole, butylated hydroxytoluene, lecithin, propyl gallate,alpha-tocopherol, and the like; and metal-chelating agents, such ascitric acid, ethylenediamine tetraacetic acid, sorbitol, tartaric acid,phosphoric acid, and the like. Coating agents for tablets, capsules,pills and like, include those used for enteric coatings, such ascellulose acetate phthalate, polyvinyl acetate phthalate, hydroxypropylmethylcellulose phthalate, hydroxypropyl methylcellulose, methacrylicacid, methacrylic acid ester copolymers, cellulose acetate trimellitate,carboxymethyl ethyl cellulose, hydroxypropyl methyl cellulose acetatesuccinate, and the like.

Pharmaceutical compositions of the invention may also be formulated toprovide slow or controlled release of the active agent using, by way ofexample, hydroxypropyl methyl cellulose in varying proportions; or otherpolymer matrices, liposomes and/or microspheres. In addition, thepharmaceutical compositions of the invention may optionally containopacifying agents and may be formulated so that they release the activeingredient only, or preferentially, in a certain portion of thegastrointestinal tract, optionally, in a delayed manner. Examples ofembedding compositions which can be used include polymeric substancesand waxes. The active agent can also be in micro-encapsulated form, ifappropriate, with one or more of the above-described excipients.

Suitable liquid dosage forms for oral administration include, by way ofillustration, pharmaceutically-acceptable emulsions, microemulsions,solutions, suspensions, syrups and elixirs. Liquid dosage formstypically comprise the active agent and an inert diluent, such as, forexample, water or other solvents, solubilizing agents and emulsifiers,such as ethyl alcohol, isopropyl alcohol, ethyl carbonate, ethylacetate, benzyl alcohol, benzyl benzoate, propylene glycol, 1,3-butyleneglycol, oils (esp., cottonseed, groundnut, corn, germ, olive, castor andsesame oils), oleic acid, glycerol, tetrahydrofuryl alcohol,polyethylene glycols and fatty acid esters of sorbitan, and mixturesthereof. Alternatively, certain liquid formulations can be converted,for example, by spray drying, to a powder, which is used to preparesolid dosage forms by conventional procedures.

Suspensions, in addition to the active ingredient, may containsuspending agents such as, for example, ethoxylated isostearyl alcohols,polyoxyethylene sorbitol and sorbitan esters, microcrystallinecellulose, aluminum metahydroxide, bentonite, agar-agar and tragacanth,and mixtures thereof.

Compound 1 can also be administered parenterally (e.g. by intravenous,subcutaneous, intramuscular or intraperitoneal injection). Forparenteral administration, the active agent is typically admixed with asuitable vehicle for parenteral administration including, by way ofexample, sterile aqueous solutions, saline, low molecular weightalcohols such as propylene glycol, polyethylene glycol, vegetable oils,gelatin, fatty acid esters such as ethyl oleate, and the like.Parenteral formulations may also contain one or more anti-oxidants,solubilizers, stabilizers, preservatives, wetting agents, emulsifiers,buffering agents, or dispersing agents. These formulations may berendered sterile by use of a sterile injectable medium, a sterilizingagent, filtration, irradiation, or heat.

Compound 1 may also be formulated as a sterile aqueous suspension orsolution for ocular injection. Useful excipients that may be included insuch an aqueous formulation include polysorbate 80, cellulose polymerssuch as carboxymethylcellulose, hydroxypropyl methylcellulose,methylcellulose, potassium chloride, calcium chloride, sodium chloride,magnesium chloride, sodium acetate, sodium citrate, histidine,α-α-trehalose dihydrate, sucrose, polysorbate 20,hydroxypropyl-β-cyclodextrin, benzalkonium chloride, Amberlite IRP-69,polyoxyethylene glycol ethers (lauryl, stearyl and oleyl),ethylenediaminetetra acetic acid sodium salt, sodium taurocholate,saponins and cremophor EL, polycarbophil-cysteine, Xanthan gum, Gellangum, hyaluronic acid, liposomes, and sodium phosphate. Permeabilityenhancers, surfactants, bile acids, cyclodextrins such as2-hydroxypropyl-β-cyclodextrin, and chelating agents may be included inthe formulation. Cylindrical oligonucleotides with a hydrophilic outersurface and a lipophilic inner surface that have the ability of formingcomplexes with an active agent may also be included in the formulation.Benzyl alcohol may serve as a preservative and sodium chloride may beincluded to adjust tonicity. In addition, hydrochloric acid and/orsodium hydroxide may be added to the solution for pH adjustment. Aqueousformulations for ocular injection may be prepared as preservative-free.

The ocular formulation may allow sustained release of the activeingredient to the eye. The ocular formulation may be formulated as anemulsion (oil in water or water in oil), a suspension, or an ointment.The suspension formulation may contain compound 1, or a pharmaceuticallyacceptable salt thereof, as a crystalline form, for example Form 1 orForm 2, or in an amorphous state.

Compound 1 may also be formulated to be suitable for eye drop dosing oras an intravitreal implant. The implant may allow delivering constanttherapeutic levels of drug. Reservoir implants are typically made with apelleted drug core surrounded by nonreactive substances such as silicon,ethylene vinyl acetate (EVA), or polyvinyl alcohol (PVA); these implantsare nonbiodegradable and can deliver continuous amounts of a drug formonths to year. Matrix implants may also be used. They are typicallyused to deliver a loading dose followed by tapering doses of the drugduring a 1-day to 6-month time period. They are most commonly made fromthe copolymers poly-lactic-acid (PLA) and/or poly-lactic-glycolic acid(PLGA), which degrade to water and carbon dioxide. Iontophoresis mayalso be used. It is a noninvasive technique in which a small electriccurrent is applied to enhance ionized drug penetration into tissue.

Encapsulated cell technology (ECT), which is a cell-based deliverysystem may also be used to deliver the therapeutic agent to the eye.Typically, genetically modified cells are packaged in a hollow tube ofsemipermeable membrane, which prevents immune-cell entry and allowsnutrients and therapeutic molecules to diffuse freely across themembrane. Two ends of the polymer section are sealed, and a titaniumloop is placed on the anchoring end, which is implanted at the parsplana and anchored to the sclera.

Compound 1 may be formulated into any form allowing delivery to the backof the eye. Examples of modes of delivery are known in the literature(Kuno et al, Polymers, 2011, 3, 193-221, del Amo et al, Drug DiscoveryToday, 2008, 13, 135-143, Short, Toxicologic Pathology, 2008, 36,49-62). Such modes of delivery include but are not limited tosuprachoroidal delivery which allows delivery to the choroid and retinathrough the suprachoroidal space, sub-Tenon delivery, peri-oculardelivery, contact lenses, punctal plugs, and scleral plugs. Compound 1may also be delivered by periocular, suprascleral, retrobulbar,peribulbar, or subconjunctival injection.

Compound 1 may be delivered as an emulsion, polymeric micro ornanospheres, liposomes, micro or nanoparticles, microspheres, micelles,or dendrimers. Biodegradable and biocompatible polymers, such aspolyactide and PLGA can be used. Compound 1 may be encapsulated.

In addition, compound 1 may be formulated for topical administration tothe skin as an ointment or cream. Ointment formulations are semisolidpreparations having a base of an oily or greasy material that istypically clear. Suitable oily materials for use in ointmentformulations include petrolatum (petroleum jelly), beeswax, cocoabutter, shea butter, and cetyl alcohol. Ointments may optionallyadditionally include emollients and penetration enhancers, if desired.

Cream formulations may be prepared as emulsions comprising an oil phaseand aqueous phase, typically including purified water. Components ofcream formulations may include: oil bases, such as petrolatrum, mineraloils, vegetable and animal oils, and triglycerides; cream bases, such aslanolin alcohols, stearic acid, and cetostearyl alcohol; a gel base,such as polyvinyl alcohol; solvents, such as, propylene glycol andpolyethylene glycol; emulsifiers, such as polysorbates, stearates, suchas glyceryl stearate, octylhydroxystearate, polyoxyl stearate, PEGstearyl ethers, isopropyl palmitate, and sorbitan monostearate;stabilizers, such as polysaccharides and sodium sulfite; emollients(i.e. moisturizers), such as medium chain triglycerides, isopropylmyristate, and dimethicone; stiffening agents, such as cetyl alcohol andstearyl alcohol; antimicrobial agents, such as methylparaben,propylparaben, phenoxyethanol, sorbic acid, diazolidinyl urea, andbutylated hydroxyanisole; penetration enhancers, such asN-methylpyrrolidone, propylene glycol, polyethylene glycol monolaurate,and the like; and chelating agents, such as edetate disodium.

Alternatively, the pharmaceutical compositions of the invention areformulated for administration by inhalation. Suitable pharmaceuticalcompositions for administration by inhalation will typically be in theform of an aerosol or a powder. Such compositions are generallyadministered using well-known delivery devices, such as a metered-doseinhaler, a dry powder inhaler, a nebulizer or a similar delivery device.

When administered by inhalation using a pressurized container, thepharmaceutical compositions of the invention will typically comprise theactive ingredient and a suitable propellant, such asdichlorodifluoromethane, trichlorofluoromethane,dichlorotetrafluoroethane, carbon dioxide or other suitable gas.Additionally, the pharmaceutical composition may be in the form of acapsule or cartridge (made, for example, from gelatin) comprisingcompound 1 and a powder suitable for use in a powder inhaler. Suitablepowder bases include, by way of example, lactose or starch.

The following non-limiting examples illustrate representativepharmaceutical compositions of the present invention.

Tablet Oral Solid Dosage Form

Compound 1, or a pharmaceutically-acceptable salt thereof is dry blendedwith microcrystalline cellulose, polyvinyl pyrrolidone, andcrosscarmellose sodium in a ratio of 4:5:1:1 and compressed into tabletsto provide a unit dosage of, for example, 5 mg, 20 mg or 40 mg activeagent per tablet.

Capsule Oral Solid Dosage Form

Compound 1, or a pharmaceutically-acceptable salt thereof is combinedwith microcrystalline cellulose, polyvinyl pyrrolidone, andcrosscarmellose sodium in a ratio of 4:5:1:1 by wet granulation andloaded into gelatin or hydroxypropyl methylcellulose capsules to providea unit dosage of, for example, 5 mg, 20 mg or 40 mg active agent percapsule.

Liquid Formulation

A liquid formulation comprising compound 1 (0.1%), water (98.9%) andascorbic acid (1.0%) is formed by adding a compound of the invention toa mixture of water and ascorbic acid.

Enteric Coated Oral Dosage Form

Compound 1 is dissolved in an aqueous solution containing polyvinylpyrrolidone and spray coated onto microcrystalline cellulose or sugarbeads in a ratio of 1:5 w/w active agent:beads and then an approximately5% weight gain of an enteric coating comprising an acrylic copolymer isapplied. The enteric coated beads are loaded into gelatin orhydroxypropyl methylcellulose capsules to provide a unit dosage of, forexample, 30 mg active agent per capsule.

Enteric Coated Oral Dosage Form

An enteric coating comprising a combination of Eudragit-L® andEudragit-S®, or hydroxypropyl methylcellulose acetate succinate isapplied to a tablet oral dosage form or a capsule oral dosage formdescribed above.

Aqueous Formulation for Ocular Injection

Each mL of a sterile aqueous suspension includes from 5 mg to 50 mg ofcompound 1, sodium chloride for tonicity, 0.99% (w/v) benzyl alcohol asa preservative, 0.75% carboxymethylcellulose sodium, and 0.04%polysorbate. Sodium hydroxide or hydrochloric acid may be included toadjust pH to 5 to 7.5.

Aqueous formulation for ocular injection A sterile preservative-freeaqueous suspension includes from 5 mg/mL to 50 mg/mL of compound 1 in 10mM sodium phosphate, 40 mM sodium chloride, 0.03% polysorbate 20, and 5%sucrose.

Ointment Formulation for Topical Administration

Compound 1 is combined with petrolatum, C₈-C₁₀ triglyceride,octylhydroxystearate, and N-methylpyrrolidone in a ratio to provide acomposition containing 0.05% to 5% active agent by weight.

Ointment Formulation for Topical Administration

Compound 1 is combined with white petrolatum, propylene glycol, mono-and di-glycerides, paraffin, butylated hydroxytoluene, and edetatecalcium disodium in a ratio to provide a composition containing 0.05% to5% active agent by weight.

Ointment Formulation for Topical Administration

Compound 1 is combined with mineral oil, paraffin, propylene carbonate,white petrolatum and white wax to provide a composition containing 0.05%to 5% active agent by weight.

Cream Formulation for Topical Administration

Mineral oil is combined with compound 1, propylene glycol, isopropylpalmitate, polysorbate 60, cetyl alcohol, sorbitan monostearate,polyoxyl 40 stearate, sorbic acid, methylparaben and propylparaben toform an oil phase, which is combined with purified water by shearblending to provide a composition containing 0.05% to 5% active agent byweight.

Cream Formulation for Topical Administration

A cream formulation comprising compound 1, benzyl alcohol, cetylalcohol, citric acid anhydrous, mono and di-glycerides, oleyl alcohol,propylene glycol, sodium cetostearyl sulphate, sodium hydroxide, stearylalcohol, triglycerides, and water contains 0.05% to 5% active agent byweight.

Cream Formulation for Topical Administration

A cream formulation comprising compound 1, cetostearyl alcohol,isopropyl myristate, propylene glycol, cetomacrogol 1000, dimethicone360, citric acid, sodium citrate, and purified water, with imidurea,methylparaben, and propylparaben, as preservatives, contains 0.05% to 5%active agent by weight.

Dry Powder Composition

Micronized compound 1 (1 g) is blended with milled lactose (25 g). Thisblended mixture is then loaded into individual blisters of a peelableblister pack in an amount sufficient to provide between about 0.1 mg toabout 4 mg of compound 1 per dose. The contents of the blisters areadministered using a dry powder inhaler.

Metered-Dose Inhaler Composition Micronized compound 1 (10 g) isdispersed in a solution prepared by dissolving lecithin (0.2 g) indemineralized water (200 mL). The resulting suspension is spray driedand then micronized to form a micronized composition comprisingparticles having a mean diameter less than about 1.5 μm. The micronizedcomposition is then loaded into metered-dose inhaler cartridgescontaining pressurized 1,1,1,2-tetrafluoroethane in an amount sufficientto provide about 0.1 mg to about 4 mg of compound 1 per dose whenadministered by the metered dose inhaler.

Nebulizer Composition

Compound 1 (25 mg) is dissolved in a solution containing 1.5-2.5equivalents of hydrochloric acid, followed by addition of sodiumhydroxide to adjust the pH to 3.5 to 5.5 and 3% by weight of glycerol.The solution is stirred well until all the components are dissolved. Thesolution is administered using a nebulizer device that provides about0.1 mg to about 4 mg of compound 1 per dose.

Utility

Compound 1 has been shown to be a potent inhibitor of the JAK family ofenzymes: JAK1, JAK2, JAK3, and TYK2.

Ocular Diseases Many ocular diseases have been shown to be associatedwith elevations of proinflammatory cytokines that rely on the JAK-STATpathway. Since compound 1 exhibits potent inhibition at all four JAKenzymes, it is expected to potently inhibit the signaling and pathogeniceffects of numerous cytokines (such as IL-6, IL-2 and IFN-γ), thatsignal through JAK, as well as to prevent the increase in othercytokines (such as MCP-1 and IP-10), whose production is driven byJAK-STAT pathway signaling.

As illustrated in the assay section, compound 1 exhibited activity incellular assays, including assays registering inhibition of thedownstream effects of cytokine elevation.

Furthermore, intravitreal dosing of compound 1 has demonstratedsignificant inhibition of IL-6 induced pSTAT3 in the rat retina/choroidtissue.

It is expected that ocular JAK inhibition in the absence of significantsystemic levels will result in potent, local anti-inflammatory activityin the eye without systemically-driven adverse effects. Compound 1 isthus expected to be beneficial in a number of ocular diseases thatinclude, but are not limited to, uveitis, diabetic retinopathy, diabeticmacular edema, dry eye disease, age-related macular degeneration,retinal vein occlusion, and atopic keratoconjunctivitis.

In particular, uveitis (Horai and Caspi, J Interferon Cytokine Res,2011, 31, 733-744), diabetic retinopathy (Abcouwer, J Clin Cell Immunol,2013, Suppl 1, 1-12), diabetic macular edema (Sohn et al., AmericanJournal of Opthalmology, 2011, 152, 686-694), dry eye disease (Stevensonet al, Arch Ophthalmol, 2012, 130, 90-100), retinal vein occlusion(Shchuko et al, Indian Journal of Ophthalmology, 2015, 63(12), 905-911)and age-related macular degeneration (Knickelbein et al, Int OphthalmolClin, 2015, 55(3), 63-78) are characterized by elevation of certainpro-inflammatory cytokines that signal via the JAK-STAT pathway.Accordingly, compound 1 is expected to be able to alleviate theassociated ocular inflammation and reverse disease progression orprovide symptom relief in these diseases.

In one aspect, therefore, the invention provides a method of treating anocular disease in a mammal, the method comprising administering apharmaceutical composition comprising compound 1, or apharmaceutically-acceptable salt thereof, and a pharmaceutical carrierto the eye of the mammal. In one aspect, the ocular disease is uveitis,diabetic retinopathy, diabetic macular edema, dry eye disease,age-related macular degeneration, retinal vein occlusion or atopickeratoconjunctivitis. In one aspect, the method comprises administeringcompound 1 by intravitreal injection.

Inflammatory Skin Disease

Inflammatory skin diseases, such as atopic dermatitis, have beenassociated with elevation of proinflammatory cytokines that rely on theJAK-STAT pathway, in particular, IL-4, IL-5, IL-10, IL-13, and IFNγ.Therefore, compound 1 is expected to be beneficial in a number dermalinflammatory or pruritic conditions that include, but are not limited toatopic dermatitis, alopecia areata, vitiligo, cutaneous T cell lymphoma,prurigo nodularis, lichen planus, primary localized cutaneousamyloidosis, bullous pemphigoid, skin manifestations of graft versushost disease, pemphigoid, discoid lupus, granuloma annulare, lichensimplex chronicus, vulvar/scrotal/perianal pruritus, lichen sclerosus,post herpetic neuralgia itch, lichen planopilaris, and foliculitisdecalvans. In particular, alopecia areata (Xing et al., Nat Med. 2014September; 20(9):1043-9), vitiligo (Craiglow et al, JAMA Dermatol. 2015October; 151(10): 1110-2), cutaneous T cell lymphoma (Netchiporouk etal., Cell Cycle. 2014; 13(21):3331-5), prurigo nodularis (Sonkoly etal., J Allergy Clin Immunol. 2006 February; 117(2):411-7), lichen planus(Welz-Kubiak et al., J Immunol Res. 2015; 2015:854747), primarylocalized cutaneous amyloidosis (Tanaka et al., Br J Dermatol. 2009December; 161(6):1217-24), bullous pemphigoid (Feliciani et al., Int JImmunopathol Pharmacol. 1999 May-August; 12(2):55-61), and dermalmanifestations of graft versus host disease (Okiyama et al., J InvestDermatol. 2014 April; 134(4):992-1000) are characterized by elevation ofcertain cytokines that signal via JAK activation. Accordingly, compound1 is expected to be able to alleviate associated dermal inflammation orpruritus driven by these cytokines.

In one aspect, therefore, the invention provides a method of treating aninflammatory skin disease in a mammal (e.g., a human), the methodcomprising applying a pharmaceutical composition comprising compound 1,or a pharmaceutically-acceptable salt thereof, and a pharmaceuticalcarrier to the skin of the mammal. In one aspect, the inflammatory skindisease is atopic dermatitis.

Compound 1 may also be used in combination with gram positiveantibiotics, such as mupirocin and fusidic acid, to treat inflammatoryskin diseases. In one aspect, therefore, the invention provides a methodof treating an inflammatory skin disease in a mammal, the methodcomprising applying compound 1 and a gram positive antibiotic to theskin of the mammal. In another aspect, the invention provides apharmaceutical composition comprising compound 1, or apharmaceutically-acceptable salt thereof, a gram positive antibiotic,and a pharmaceutically-acceptable carrier.

Respiratory Diseases

Cytokines which signal through the JAK-STAT pathway, in particular IL-2,IL-3, IL-4, IL-5, IL-6, IL-9, IL-11, IL-13, IL-23, IL-31, IL-27, thymicstromal lymphopoietin (TSLP), interferon-γ (IFNγ) andgranulocyte-macrophage colony-stimulating factor (GM-CSF) have also beenimplicated in asthma inflammation and in other inflammatory respiratorydiseases. As described above, compound 1 has been shown to be a potentinhibitor of the JAK1, JAK2, JAK3, and TYK2 enzymes and has alsodemonstrated potent inhibition of pro-inflammatory cytokines in cellularassays.

The anti-inflammatory activity of JAK inhibitors has been robustlydemonstrated in preclinical models of asthma (Malaviya et al., IntImmunopharmacol, 2010, 10, 829-836; Matsunaga et al., Biochem andBiophys Res Commun, 2011, 404, 261-267; Kudlacz et al., Eur J Pharmacol,2008, 582, 154-161.) Accordingly, compound 1 is expected to be usefulfor the treatment of inflammatory respiratory disorders, in particular,asthma. Inflammation and fibrosis of the lung is characteristic of otherrespiratory diseases in addition to asthma such as chronic obstructivepulmonary disease (COPD), cystic fibrosis (CF), pneumonitis,interstitial lung diseases (including idiopathic pulmonary fibrosis),acute lung injury, acute respiratory distress syndrome, bronchitis,emphysema, and bronchiolitis obliterans. Compound 1, therefore, is alsoexpected to be useful for the treatment of chronic obstructive pulmonarydisease, cystic fibrosis, pneumonitis, interstitial lung diseases(including idiopathic pulmonary fibrosis), acute lung injury, acuterespiratory distress syndrome, bronchitis, emphysema, bronchiolitisobliterans, and sarcoidosis.

In one aspect, therefore, the invention provides a method of treating arespiratory disease in a mammal (e.g., a human), the method comprisingadministering to the mammal compound 1, or a pharmaceutically-acceptablesalt thereof.

In one aspect, the respiratory disease is asthma, chronic obstructivepulmonary disease, cystic fibrosis, pneumonitis, chronic obstructivepulmonary disease (COPD), cystic fibrosis (CF), pneumonitis,interstitial lung diseases (including idiopathic pulmonary fibrosis),acute lung injury, acute respiratory distress syndrome, bronchitis,emphysema, bronchiolitis obliterans, or sarcoidosis. In another aspect,the respiratory disease is asthma or chronic obstructive pulmonarydisease.

In a further aspect, the respiratory disease is a lung infection, ahelminthic infection, pulmonary arterial hypertension, sarcoidosis,lymphangioleiomyomatosis, bronchiectasis, or an infiltrative pulmonarydisease. In yet another aspect, the respiratory disease is drug-inducedpneumonitis, fungal induced pneumonitis, allergic bronchopulmonaryaspergillosis, hypersensitivity pneumonitis, eosinophilic granulomatosiswith polyangiitis, idiopathic acute eosinophilic pneumonia, idiopathicchronic eosinophilic pneumonia, hypereosinophilic syndrome, Lifflersyndrome, bronchiolitis obliterans organizing pneumonia, orimmune-checkpoint-inhibitor induced pneumonitis.

The invention further provides a method of treating asthma in a mammal,the method comprising administering to the mammal a pharmaceuticalcomposition comprising compound 1, or a pharmaceutically-acceptable saltthereof and a pharmaceutically-acceptable carrier.

Compound 1, or a pharmaceutically acceptable salt thereof, is alsoexpected to be useful to treat eosinophilic lung diseases. Eosinophilicairway inflammation which is a characteristic feature of diseasescollectively termed eosinophilic lung diseases (Cottin et al., Clin.Chest. Med., 2016, 37(3), 535-56). Eosinophilic diseases have beenassociated with IL-4, IL-13 and IL-5 signaling. Eosinophilic lungdiseases include infections (especially helminthic infections),drug-induced pneumonitis (induced for example by therapeutic drugs suchas antibiotics, phenytoin, or 1-tryptophan), fungal-induced pneumonitis(e.g. allergic bronchopulmonary aspergillosis), hypersensitivitypneumonitis and eosinophilic granulomatosis with polyangiitis (formerlyknown as Churg-Strauss syndrome). Eosinophilic lung diseases of unknownetiology include idiopathic acute eosinophilic pneumonia, idiopathicchronic eosinophilic pneumonia, hypereosinophilic syndrome, and Lifflersyndrome.

Compound 1, or a pharmaceutically acceptable salt thereof, may also beuseful to treat PAH. A polymorphism in the IL-6 gene has been associatedwith elevated IL-6 levels and an increased risk of developing pulmonaryarterial hypertension (PAH) (Fang et al., J Am Soc Hypertens., 2017,11(3), 171-177). Corroborating the role of IL-6 in PAH, inhibition ofthe IL-6 receptor chain gp130 ameliorated the disease in a rat model ofPAH (Huang et al., Can J Cardiol., 2016, 32(11), 1356.e1-1356.e10).

Compound 1, or a pharmaceutically acceptable salt thereof, may also beuseful to treat non-allergic lung diseases such as sarcoidosis, andlymphangioleiomyomatosis. Cytokines such as IFNγ, IL-12 and IL-6 havebeen implicated in a range of non-allergic lung diseases such assarcoidosis, and lymphangioleiomyomatosis (El-Hashemite et al., Am. J.Respir. Cell Mol. Biol., 2005, 33, 227-230, and El-Hashemite et al.,Cancer Res., 2004, 64, 3436-3443).

Compound 1, or a pharmaceutically acceptable salt thereof, may also beuseful to treat bronchiectasis and infiltrative pulmonary diseases whichare diseases associated with chronic neutrophilic inflammation. Certaincytokines are associated with neutrophilic inflammation (e.g. IL-6,IFNγ).

Pathological T cell activation is critical in the etiology of multiplerespiratory diseases. Autoreactive T cells play a role in bronchiolitisobliterans organizing pneumonia (also termed COS). Similar to COS theetiology of lung transplant rejections is linked to an aberrant T cellactivation of the recipients T cells by the transplanted donor lung.Lung transplant rejections may occur early as Primary Graft Dysfunction(PGD), organizing pneumonia (OP), acute rejection (AR) or lymphocyticbronchiolitis (LB) or they may occur years after lung transplantation asChronic Lung Allograft Dysfunction (CLAD). CLAD was previously known asbronchiolitis obliterans (BO) but now is considered a syndrome that canhave different pathological manifestations including BO, restrictiveCLAD (rCLAD or RAS) and neutrophilic allograft dysfunction. Chronic lungallograft dysfunction (CLAD) is a major challenge in long-termmanagement of lung transplant recipients as it causes a transplantedlung to progressively lose functionality (Gauthier et al., CurrTransplant Rep., 2016, 3(3), 185-191). CLAD is poorly responsive totreatment and therefore, there remains a need for effective compoundscapable of preventing or treating this condition. Several JAK-dependentcytokines such as IFNγ and IL-5 are up-regulated in CLAD and lungtransplant rejection (Berastegui et al, Clin Transplant. 2017, 31,e12898). Moreover, high lung levels of CXCR3 chemokines such as CXCL9and CXCL10 which are downstream of JAK-dependent IFN signaling, arelinked to worse outcomes in lung transplant patients (Shino et al, PLOSOne, 2017, 12 (7), e0180281). JAK inhibition has been shown to beeffective in kidney transplant rejection (Vicenti et al., AmericanJournal of Transplantation, 2012, 12, 2446-56). Therefore, compound 1has the potential to be effective in treating or preventing lungtransplant rejection and CLAD. Similar T cell activation events asdescribed as the basis for lung transplant rejection also are consideredthe main driver of lung graft-versus-host disease (GVHD) which can occurpost hematopoietic stem cell transplants. Similar to CLAD, lung GVHD isa chronic progressive condition with extremely poor outcomes and notreatments are currently approved. A retrospective, multicenter surveystudy of 95 patients with steroid-refractory acute or chronic GVHD whoreceived the systemic JAK inhibitor ruxolitinib as salvage therapydemonstrated complete or partial response to ruxolitinib in the majorityof patients including those with lung GVHD (Zeiser et al, Leukemia,2015, 29, 10, 2062-68). More recently, immune-checkpoint inhibitorinduced pneumonitis, another T cell mediated lung disease emerged withthe increased use of immune-checkpoint inhibitors. In cancer patientstreated with these T cell stimulating agents, fatal pneumonitis candevelop. Compound 1, or a pharmaceutically acceptable salt thereof, hasthe potential to present a novel treatment for these underserved seriousrespiratory diseases.

Gastrointestinal Diseases

As a JAK inhibitor, compound 1, or a pharmaceutically acceptable saltthereof, may also be useful for a variety of other diseases. Compound 1,or a pharmaceutically acceptable salt thereof, may be useful for avariety of gastrointestinal inflammatory indications that include, butare not limited to, inflammatory bowel disease, ulcerative colitis(proctosigmoiditis, pancolitis, ulcerative proctitis and left-sidedcolitis), Crohn's disease, collagenous colitis, lymphocytic colitis,Behcet's disease, celiac disease, immune checkpoint inhibitor inducedcolitis, ileitis, eosinophilic esophagitis, graft versus hostdisease-related colitis, and infectious colitis. Ulcerative colitis(Reimund et al., J Clin Immunology, 1996, 16, 144-150), Crohn's disease(Woywodt et al., Eur J Gastroenterology Hepatology, 1999, 11, 267-276),collagenous colitis (Kumawat et al., Mol Immunology, 2013, 55, 355-364),lymphocytic colitis (Kumawat et al., 2013), eosinophilic esophagitis(Weinbrand-Goichberg et al., Immunol Res, 2013, 56, 249-260), graftversus host disease-related colitis (Coghill et al., Blood, 2001, 117,3268-3276), infectious colitis (Stallmach et al., Int J Colorectal Dis,2004, 19, 308-315), Behcet's disease (Zhou et al., Autoimmun Rev, 2012,11, 699-704), celiac disease (de Nitto et al., World J Gastroenterol,2009, 15, 4609-4614), immune checkpoint inhibitor induced colitis (e.g.,CTLA-4 inhibitor-induced colitis; (Yano et al., J Translation Med, 2014,12, 191), PD-1- or PD-Li-inhibitor-induced colitis), and ileitis(Yamamoto et al., Dig Liver Dis, 2008, 40, 253-259) are characterized byelevation of certain pro-inflammatory cytokine levels. As manypro-inflammatory cytokines signal via JAK activation, compound 1, or apharmaceutically acceptable salt thereof, may be able to alleviate theinflammation and provide symptom relief. In particular, compound 1, or apharmaceutically acceptable salt thereof may be useful for the inductionand maintenance of remission of ulcerative colitis, and for thetreatment of Crohn's disease, immune checkpoint inhibitor inducedcolitis, and the gastrointestinal adverse effects in graft versus hostdisease. In one aspect, therefore, the invention provides a method oftreating a gastrointestinal inflammatory disease in a mammal (e.g., ahuman), the method comprising administering to the mammal, compound 1,or a pharmaceutically acceptable salt thereof, or a pharmaceuticalcomposition comprising a pharmaceutically-acceptable carrier andcompound 1, or a pharmaceutically acceptable salt thereof.

Other Diseases

Compound 1, or a pharmaceutically acceptable salt thereof, may also beuseful to treat other diseases such as other inflammatory diseases,autoimmune diseases or cancers.

Compound 1, or a pharmaceutically acceptable salt thereof, may be usefulto treat one or more of arthritis, rheumatoid arthritis, juvenilerheumatoid arthritis, transplant rejection, xerophthalmia, psoriaticarthritis, diabetes, insulin dependent diabetes, motor neurone disease,myelodysplastic syndrome, pain, sarcopenia, cachexia, septic shock,systemic lupus erythematosus, leukemia, chronic lymphocytic leukemia,chronic myelocytic leukemia, acute lymphoblastic leukemia, acutemyelogenous leukemia, ankylosing spondylitis, myelofibrosis, B-celllymphoma, hepatocellular carcinoma, Hodgkins disease, breast cancer,Multiple myeloma, melanoma, non-Hodgkin lymphoma, non-small-cell lungcancer, ovarian clear cell carcinoma, ovary tumor, pancreas tumor,polycythemia vera, Sjoegrens syndrome, soft tissue sarcoma, sarcoma,splenomegaly, T-cell lymphoma, and thalassemia major.

Combination Therapy

Compound 1, or a pharmaceutically acceptable salt thereof may be used incombination with one or more agents which act by the same mechanism orby different mechanisms to treat a disease. The different agents may beadministered sequentially or simultaneously, in separate compositions orin the same composition. Useful classes of agents for combinationtherapy include, but are not limited to, anti-angiogenic, steroid,anti-inflammatory, plasma kallikrein inhibitor, placenta growth factorligand inhibitor, VEGF-A ligand inhibitor, angiopoietin ligand-2inhibitor, protein tyrosine phosphatase beta inhibitor, Tek tyrosinekinase receptor stimulator, calcineurin inhibitor, VEGF ligandinhibitor, mTOR complex 1 inhibitor, mTOR inhibitor, IL-17 antagonist,calmodulin modulator, FGF receptor antagonist, PDGF receptor antagonist,VEGF receptor antagonist, TNF alpha ligand inhibitor, TNF binding agent,proteoglycan 4 stimulator, VEGF-C ligand inhibitor, VEGF-D ligandinhibitor, CD126 antagonist, complement cascade inhibitor,glucocorticoid agonist, complement C5 factor inhibitor, cannabinoidreceptor antagonist, sphingosine-1-phosphate receptor-1 modulator,sphingosine-1-phosphate receptor-3 modulator, sphingosine-1-phosphatereceptor-4 modulator, sphingosine-1-phosphate receptor-5 modulator,acetaldehyde dehydrogenase inhibitor, Flt3 tyrosine kinase inhibitor,Kit tyrosine kinase inhibitor, Protein kinase C inhibitor,adrenocorticotrophic hormone ligand, stromal cell-derived factor 1ligand inhibitor, immunoglobulin G1 agonist; Interleukin-1 beta ligandinhibitor, mucin stimulator; Nuclear factor kappa B modulator, cytotoxicT-lymphocyte protein-4 stimulator, T cell surface glycoprotein CD28inhibitor, lipoprotein lipase stimulator; PPAR alpha agonist, adenosineA3 receptor agonist, angiotensin II receptor antagonist, VEGF receptorantagonist, interferon beta ligand, SMAD-2 modulator; TGF beta 1 ligandinhibitor, somatostatin receptor agonist, IL-2 receptor alpha subunitinhibitor, VEGF-B ligand inhibitor, thymosin beta 4 ligand, angiotensinII AT-1 receptor antagonist, CCR2 chemokine antagonist, membrane copperamine oxidase inhibitor, CD11a antagonist, ICAM-1 inhibitor,insulin-like growth factor 1 antagonist, kallikrein inhibitor,fucosyltransferase 6 stimulator, GDP fucose synthetase modulator, GHRgene inhibitor, IGF1 gene inhibitor, VEGF-1 receptor antagonist, albuminagonist, IL-2 antagonist, CSF-1 antagonist; PDGF receptor antagonist,VEGF-2 receptor antagonist, mTOR inhibitor, PPAR alpha agonist, RhoGTPase inhibitor, Rho associated protein kinase inhibitor, complement C3inhibitor, EGR-1 transcription factor inhibitor, nuclear erythroid2-related factor modulator, nuclear factor kappa B inhibitor, integrinalpha-V/beta-3 antagonist, erythropoietin receptor agonist,glucagon-like peptide 1 agonist, TNFRSF1A gene stimulator, angiopoietinligand-2 inhibitor, alpha-2 antiplasmin inhibitor, collagen antagonist,fibronectin inhibitor, laminin antagonist, plasmin stimulator, nervegrowth factor ligand, FGF1 receptor antagonist, FGF3 receptorantagonist, itk tyrosine kinase inhibitor, Lck tyrosine kinaseinhibitor, Ltk tyrosine kinase receptor inhibitor, PDGF receptor alphaantagonist, PDGF receptor beta antagonist, protein tyrosine kinaseinhibitor, VEGF-3 receptor antagonist, membrane copper amine oxidaseinhibitor, somatostatin 2 receptor agonist, somatostatin 4 receptoragonist, somatostatin 5 receptor agonist, protein kinase C alphainhibitor, protein kinase C beta inhibitor, protein kinase C deltainhibitor protein kinase C epsilon inhibitor protein kinase C etainhibitor, protein kinase C theta inhibitor, ankyrin modulator, mucinstimulator, P2Y2 purinoceptor agonist, gap junction alpha-1 proteininhibitor, CCR3 chemokine antagonist; eotaxin ligand inhibitor,amiloride sensitive sodium channel inhibitor, PDGF receptor antagonist,protein tyrosine kinase inhibitor, retinal pigment epithelium proteininhibitor, matrix metalloprotease inhibitor, PDGF receptor antagonist,PDGF receptor beta antagonist, PDGF-B ligand inhibitor, growth hormonereceptor antagonist, cell adhesion molecule inhibitor, integrinmodulator, CXCR4 chemokine antagonist, coiled coil domain containingprotein inhibitor, Hsp 90 modulator, Rho associated protein kinaseinhibitor, VEGF gene inhibitor, endoglin inhibitor, CCR3 chemokineantagonist, maxi K potassium channel modulator, maxi K potassium channelstimulator, PGF2 alpha agonist, prostanoid receptor agonist, voltagegated chloride channel 2 modulator, complement C5a receptor antagonist,inosine monophosphate dehydrogenase inhibitor, interleukin 18 ligandinhibitor, TRP cation channel M8 stimulator, CNTF receptor agonist,TRPV1 gene inhibitor, deoxyribonuclease I stimulator, IRS 1 geneinhibitor, Rho associated protein kinase inhibitor, poly ADP ribosepolymerase 1 inhibitor, poly ADP ribose polymerase 2 inhibitor, poly ADPribose polymerase 3 inhibitor, vanilloid VR1 agonist, NFAT5 genestimulator, Mucin stimulator, Syk tyrosine kinase inhibitor, alpha 2adrenoceptor agonist, cyclooxygenase inhibitor, amyloid proteindeposition inhibitor, glycogen synthase kinase-3 inhibitor, PARPstimulator, tau deposition inhibitor, DDIT4 gene inhibitor, hemoglobinsynthesis modulator, interleukin-1 beta ligand inhibitor, TNFantagonist, KCNQ voltage-gated potassium channel stimulator, NMDAreceptor antagonist, cyclooxygenase 1 inhibitor, cyclooxygenaseinhibitor, 5-HT 1a receptor agonist, calcium channel inhibitor, FGF-2ligand modulator, phosphoinositide 3-kinase inhibitor, CD44 antagonist,hyaluronidase modulator, hyaluronic acid agonist, IL-1 antagonist, typeI IL-1 receptor antagonist, complement factor P inhibitor, tubulinantagonist, beta amyloid antagonist, IL2 gene stimulator, I-kappa Bkinase beta inhibitor, nuclear factor kappa B modulator, plasminogenactivator inhibitor 1 inhibitor, FGF-2 ligand, protease modulator, andcorticotropin modulator.

Specific agents that may be used in combination with compound 1 include,but are not limited to lanadelumab, aflibercept, RG-7716, AKB-9778,ciclosporin, bevacizumab, everolimus, secukinumab, fluocinoloneacetonide, RP-101, squalamine lactate, recombinant human lubricin,OPT-302, sarilumab, dexamethasone, eculizumab, fingolimod, adalimumab,reproxalap, midostaurin, corticotropin, olaptesed pegol, canakinumab,recoflavone, abatacept, fenofibrate, piclidenoson, OpRegen, candesartan,golimumab, pegaptanib, interferon-beta, disitertide, octreotide acetate,anecortave, basiliximab, suprachoroidal triamcinolone acetonide,RGN-259, difluprednate, HL-036, avacincaptad pegol sodium, irbesartan,propagermanium, triamcinolone acetonide, azithromycin, BI-1467335,lifitegrast, loteprednol etabonate, teprotumumab, KVD-001, TZ-101,atesidorsen, Nov-03, bevacizumab, AVA-101, RU-101, voclosporin,vorolanib, sirolimus, choline fenofibrate, VX-210, APL-2, CPC-551,elamipretide, SF-0166, cibinetide, elamipretide, liraglutide, EYS-606,nesvacumab, aflibercept, ocriplasmin, filgotinib, cenegermin, adipocell,brolucizumab, ranibizumab, aflibercept, padeliporfin photodynamictherapy, pazopanib, ASP-8232, veldoreotide, sotrastaurin, abiciparpegol, diquafosol tetrasodium, HCB-1019, conbercept, bertilimumab,SHP-659, THR-317, ALK-001, PAN-90806, interferon alfa-2b, fluocinolone,sunitinib malate, emixustat, hI-conl, TB-403, minocycline, MA09-hRPEcells, pegpleranib sodium, pegvisomant, luminate, burixafor, H-1129,carotuximab, AXP-1275, ranibizumab, isopropyl unoprostone, tesidolumab,enteric-coated mycophenolate sodium, tadekinig alfa, triamcinoloneacetonide, cyclosporine, ST-266, AVX-012, NT-501-ECT, tivanisiran,verteporfin, dornase alfa, aganirsen, ripasudil, rucaparib phosphate,zucapsaicin, tetrathiomolybdate, diclofenac, LHA-510, AGN-195263,tacrolimus, rebamipide, R-348, brimonidine tartrate, vizomitin, T-89,LME-636, BI-1026706, rimexolone, tobramycin, TOP-1630, talaporfin,bromfenac sodium, triamcinolone acetonide, davunetide, loteprednoletabonate, XED-60, EG-Mirotin, APD-209, adenovir, PF-04523655,hydroxycarbamide, navamepent, retinalamin, CNTO-2476, ranibizumab,flupirtine, B27PD, S-646240, GLY-230, hydralazine, nepafenac, DexNP,Trehalose, hyaluronic acid, dexamethasone-Ca sustained-release depot,naluzotan, hyaluronidase, sodium hyaluronate, isunakinra, somatostatin,CLG-561, OC-10X, UCA-002, recombinant human epidermal growth factor,pemirolast, VM-100, MB-11316, monosodium alpha luminol, ranibizumab,IMD-1041, LMG-324, HE-10, cinhyaluronate sodium, BDM-E, mesenchymalprecursor cells, disulfiram, CTC-96, PG-101, Beifushu, chymotrypsin.

Also provided, herein, is a pharmaceutical composition comprisingcompound 1, or a pharmaceutically acceptable salt thereof, and one ormore other therapeutic agents. The therapeutic agent may be selectedfrom the class of agents specified above and from the list of specificagent described above. In some embodiments, the pharmaceuticalcomposition is suitable for ocular delivery. In some embodiments, thepharmaceutical composition is a liquid or suspension composition.

Further, in a method aspect, the invention provides a method of treatinga disease or disorder in a mammal comprising administering to the mammalcompound 1, or a pharmaceutically acceptable salt thereof, and one ormore other therapeutic agents.

When used in combination therapy, the agents may be formulated in asingle pharmaceutical composition, or the agents may be provided inseparate compositions that are administered simultaneously or atseparate times, by the same or by different routes of administration.Such compositions can be packaged separately or may be packaged togetheras a kit. The two or more therapeutic agents in the kit may beadministered by the same route of administration or by different routesof administration.

Examples

The following synthetic and biological examples are offered toillustrate the invention, and are not to be construed in any way aslimiting the scope of the invention.

In the examples below, the following abbreviations have the followingmeanings unless otherwise indicated. Abbreviations not defined belowhave their generally accepted meanings.

-   ACN=acetonitrile-   Boc=tert-butoxycarbonyl-   DCC=dicyclohexylcarbodiimide-   DIPEA=N,N-diisopropylethylamine-   DMAc=dimethylacetamide-   DMF=N,N-dimethylformamide-   DMSO=dimethyl sulfoxide-   EtOAc=ethyl acetate-   HATU=N,N,N′,N′-tetramethyl-O-(7-azabenzotriazol-1-yl)uronium    hexafluorophosphate-   LDA=lithium diisopropylamide-   min=minute(s)-   MTBE=methyl tert-butyl ether-   NBS=N-bromosuccinimide-   NMP=N-Methyl-2-pyrrolidone-   RT=room temperature-   THF=tetrahydrofuran-   bis(pinacolato)diboron=4,4,5,5,4′,4′,5′,5′-octamethyl-[2,2′]bi[[1,3,2]dioxaborolanyl]-   Pd(dppf)Cl₂—CH₂Cl₂=dichloro(1,1′-bis(diphenylphosphino)-ferrocene)-dipalladium(II)    complex with dichloromethane

Reagents and solvents were purchased from commercial suppliers (Aldrich,Fluka, Sigma, etc.), and used without further purification. Progress ofreaction mixtures was monitored by thin layer chromatography (TLC),analytical high performance liquid chromatography (anal. HPLC), and massspectrometry. Reaction mixtures were worked up as described specificallyin each reaction; commonly they were purified by extraction and otherpurification methods such as temperature-, and solvent-dependentcrystallization, and precipitation. In addition, reaction mixtures wereroutinely purified by column chromatography or by preparative HPLC,typically using C18 or BDS column packings and conventional eluents.Typical preparative HPLC conditions are described below.

Characterization of reaction products was routinely carried out by massand ¹H-NMR spectrometry. For NMR analysis, samples were dissolved indeuterated solvent (such as CD₃OD, CDCl₃, or d₆-DMSO), and ¹H-NMRspectra were acquired with a Varian Gemini 2000 instrument (400 MHz)under standard observation conditions. Mass spectrometric identificationof compounds was performed by an electrospray ionization method (ESMS)with an Applied Biosystems (Foster City, Calif.) model API 150 EXinstrument or a Waters (Milford, Mass.) 3100 instrument, coupled toautopurification systems.

Preparative HPLC Conditions Column: C18, 5 μm. 21.2×150 mm or C18, 5 μm21×250 or C14, 5 μm 21×150 mm

Column temperature: Room TemperatureFlow rate: 20.0 mL/min

Mobile Phases:

-   -   A=Water+0.05% TFA    -   B=ACN+0.05% TFA,        Injection volume: (100-1500 μL)        Detector wavelength: 214 nm

Crude compounds were dissolved in 1:1 water:acetic acid at about 50mg/mL. A 4 minute analytical scale test run was carried out using a2.1×50 mm C18 column followed by a 15 or 20 minute preparative scale runusing 100 μL injection with the gradient based on the % B retention ofthe analytical scale test run. Exact gradients were sample dependent.Samples with close running impurities were checked with a 21×250 mm C18column and/or a 21×150 mm C14 column for best separation. Fractionscontaining desired product were identified by mass spectrometricanalysis.

Preparation 1:2-(4-(benzyloxy)-2-ethyl-5-fluorophenyl)-4,4,5,5-tetramethyl-1,3,2-dioxaborolane(1-5)

(a) 2-(Benzyloxy)-4-bromo-1-fluorobenzene (1-2)

Two reactions were carried out in parallel and combined for work-up. Amixture of 5-bromo-2-fluorophenol (1-1) (850 g, 4.5 mol), benzyl bromide(837 g, 4.9 mol) and potassium carbonate (923 g, 6.7 mol) in ACN (5 L)was stirred at 20° C. for 12 h. The reactions were combined andconcentrated, diluted with water (8 L), and extracted with EtOAc (3×3L). The organic layer was separated, washed with brine (3 L), dried oversodium sulfate and concentrated. The crude product was purified througha silica gel pad (eluted with 3:1 petroleum ether:EtOAc) to give thetitle intermediate (1.83 kg, 73% yield) as a white solid. ¹H NMR (400MHz, CDCl₃) δ 7.38-7.46 (m, 5H), 7.15 (dd, J=7.6, 2.0 Hz, 1H), 6.98-7.15(m, 1H), 5.12 (s, 2H).

(b) 2-(Benzyloxy)-4-ethyl-1-fluorobenzene (1-3)

Six reactions were carried out in parallel and combined for work-up. Toa solution of the product of the previous step (200 g, 711 mmol) in THF(100 mL) was added potassium carbonate (197 g, 1.4 mol). The reactionmixture was purged with nitrogen 3 times, followed by addition ofPd(dppf)Cl₂—CH₂Cl₂ (11.6 g, 14.2 mmol). The reaction mixture was cooledto 0° C., diethylzinc (1 M, 1.07 L) was added drop-wise, and thereaction mixture was stirred at 70° C. for 1 h. The reactions werecombined, cooled to 20° C. and poured into water (7 L) slowly. To themixture was added aq. 4 M HCl to pH 6. The organic layer was separated,and the aqueous phase was extracted with EtOAc (3×2 L). The combinedorganic layer was washed with brine (5 L), dried over sodium sulfate,concentrated, and purified through a silica gel pad (eluted with 50:1petroleum ether:EtOAc)) to give the title intermediate (900 g, 92%yield) as a light yellow oil. ¹H NMR (400 MHz, CDCl₃) δ 7.29-7.43 (m,5H), 6.94-6.97 (m, 1H), 6.82 (d, J=8.0 Hz, 1H), 6.70 (m, 1H), 5.09 (s,2H), 2.52-2.58 (m, 2H), 1.17 (t, J=7.6 Hz, 3H).

(c) 1-(Benzyloxy)-4-bromo-5-ethyl-2-fluorobenzene (1-4)

Four reactions were carried out in parallel and combined. To a solutionof 2-(benzyloxy)-4-ethyl-1-fluorobenzene (1-3) (293 g, 1.3 mol) in ACN(1 L) was added NBS (249 g, 1.4 mol) in portions at 20° C. The reactionmixture was stirred at 20° C. for 2 h. The reaction mixtures werecombined and concentrated. The residue was diluted with water (5 L) andextracted with EtOAc (2×5 L). The organic phase was washed with brine (4L), dried over anhydrous sodium sulfate, filtered and concentrated invacuo. The crude product was purified by silica gel chromatography(eluted with petroleum ether:EtOAc 100:1-10:1) to give the titleintermediate (1.4 kg, 89% yield) as light yellow oil. ¹H NMR (400 MHz,CDCl₃) δ 7.29-7.38 (m, 5H), 7.2 (d, J=10.4 Hz, 1H), 6.8 (d, J=8.8 Hz,1H), 5.06 (s, 2H), 2.6 (q, J=7.6 Hz, 2H), 1.1 (t, J=7.6 Hz, 3H).

(d)2-(4-(benzyloxy)-2-ethyl-5-fluorophenyl)-4,4,5,5-tetramethyl-1,3,2-dioxaborolane(1-5)

Seven reactions were carried out in parallel and combined for work-up.To a solution of the product of the previous step (200 g, 647 mmol) indioxane (2 L) was added potassium acetate (190 g, 1.9 mol),bis(pinacolato)diboron (181 g, 712 mmol), and Pd(dppf)Cl₂—CH₂Cl₂ (10.6g, 12.9 mmol) under nitrogen at 20° C. The mixture was stirred at 120°C. for 2 h. The reaction mixtures were combined, concentrated, dilutedwith water (5 L), and extracted with EtOAc (3×4 L). The combined organicphase was dried with anhydrous sodium sulfate, filtered and concentratedin vacuo. The crude product was purified by silica gel chromatography(eluted with petroleum ether:EtOAc 1:0-5:1) to give the title compound(1.35 kg, 84% yield) as a white solid. ¹H NMR (400 MHz, CDCl₃) δ7.33-7.51 (m, 6H), 6.82 (d, J=7.6 Hz, 1H), 5.17 (s, 2H), 2.85 (q, J=7.6Hz, 2H), 1.33 (s, 12H), 1.15 (t, J=7.6 Hz, 3H).

Preparation 2: 1-Benzyl-4-imino-1,4-dihydropyridin-3-amine (2)

To a solution of pyridine-3,4-diamine (400 g, 3.67 mol) in ACN (3 L) wasadded benzyl bromide (596 g, 3.49 mol) in portions at 0° C. and thereaction mixture was stirred for 30 min and then at 20° C. for 12 h, andfiltered. The filter cake was washed with ACN (500 mL) and dried to givethe HBr salt of the title compound (600 g, 2.14 mol, 58% yield) as awhite powder. ¹H NMR (400 MHz, MeOD) δ 7.83 (d, J=5.6 Hz, 1H), 7.64 (s,1H), 7.32-7.40 (m, 5H), 6.76 (d, J=6.8 Hz, 1H), 5.28 (s, 2H).

Preparation 3:6-(4-(Benzyloxy)-2-ethyl-5-fluorophenyl)-4-fluoro-1H-indazole-3-carbaldehyde(3)

(a) 1-(4-Bromo-2,6-difluorophenyl)-2,2-diethoxyethan-1-one (3-1)

Nine reactions were carried out in parallel and combined for work-up. Asolution of 1-bromo-3,5-difluorobenzene (100 g, 518 mmol) in THF (700mL) was degassed and purged with nitrogen three times. Then 2 M LDA (311mL) was added at −70° C. and the reaction mixture was stirred at −70° C.for 0.5 h under nitrogen. A solution of ethyl 2,2-diethoxyacetate (96 g,544 mmol) in THF (200 mL) was added drop-wise at −70° C. under nitrogenand the reaction mixture was stirred for 1 h. The reactions werecombined and poured into ice saturated ammonium chloride (10 L) inportions and extracted with EtOAc (3×3 L). The organic layer wasseparated, washed with brine (5 L), dried over sodium sulfate,concentrated, and purified by silica gel chromatography (eluted withpetroleum ether EtOAc 1:0-100:1) to give the title compound (1.26 kg,84% yield) as a yellow oil. ¹H NMR (400 MHz, CDCl₃) δ 7.12 (d, J=7.2 Hz,2H), 5.15 (s, 1H), 3.61-3.7 (m, 4H), 1.2 (t, J=7.2 Hz, 6H).

(b)1-(4′-(benzyloxy)-2′-ethyl-3,5,5′-trifluoro-[1,1′-biphenyl]-4-yl)-2,2-diethoxyethan-1-one(3-2)

Five reactions were carried out in parallel and combined for work-up. Toa mixture of 1-(4-bromo-2,6-difluorophenyl)-2,2-diethoxyethan-1-one(3-1) (189 g, 586 mmol) in ethanol (150 mL) and toluene (1.5 L) wasadded water (150 mL), sodium carbonate (84.8 g, 800 mmol), and2-(4-(benzyloxy)-2-ethyl-5-fluorophenyl)-4,4,5,5-tetramethyl-1,3,2-dioxaborolane(1-5) (190 g, 533 mmol) at 20° C. The suspension was degassed undervacuum and purged with nitrogen several times. Pd(dppf)Cl₂—CH₂Cl₂ (13 g,16 mmol) was added and the reaction mixture was purged with nitrogenseveral times and stirred at 120° C. for 2 h. The reactions werecombined, cooled to 20° C., poured into water (5 L) and extracted withEtOAc (3×4 L). The combined organic layers were washed with brine (5 L),dried over sodium sulfate, filtered, concentrated, and purified bysilica gel chromatography (eluted with petroleum ether:EtOAc 100:1-5:1)to give the title intermediate (880 g, 70% yield) as a yellow oil. ¹HNMR (400 MHz, CDCl₃) δ 7.36-7.48 (m, 5H), 6.94-6.96 (m, 2H), 6.86-6.92(m, 2H), 5.29 (s, 1H), 5.19 (s, 2H), 3.67-3.77 (m, 4H), 2.52 (q, J=7.6Hz, 2H), 1.25 (t, J=6.8 Hz, 6H), 1.07 (t, J=7.2 Hz, 3H).

(c)6-(4-(benzyloxy)-2-ethyl-5-fluorophenyl)-3-(diethoxymethyl)-4-fluoro-1H-indazole(3-3)

Four reactions were carried out in parallel and combined for work-up. Toa solution of the product of the previous step (220 g, 466 mmol) in THF(2 L) was added hydrazine monohydrate (47.6 g, 931 mmol) at 20° C. Thereaction mixture was stirred at 100° C. for 12 h. Four reactions werecombined and cooled to 20° C. and concentrated. The residue wasdissolved in EtOAc (5 L) and washed with 0.1 M HCl (2×1.5 L). Thecombined organic layers were washed with brine (1.5 L), dried oversodium sulfate, filtered and concentrated to give the title intermediate(900 g, crude) as yellow gum, which was used directly in the next step.¹H NMR (400 MHz, CDCl₃) δ 7.36-7.48 (m, 5H), 6.94-6.96 (m, 2H),6.86-6.92 (m, 2H), 5.29 (s, 1H), 5.19 (s, 2H), 3.67-3.77 (m, 4H), 2.52(q, J=7.6 Hz, 2H), 1.25 (t, J=6.8 Hz, 6H), 1.07 (t, J=7.2 Hz, 3H).

(d)6-(4-(Benzyloxy)-2-ethyl-5-fluorophenyl)-4-fluoro-1H-indazole-3-carbaldehyde(3)

Three reactions were carried out in parallel and combined for work-up.To a solution of the product of the previous step (300 g, 643 mmol) inacetone (1.5 L) was added 4 M HCl (16 mL) drop-wise at 20° C. and thereaction mixture was stirred at 20° C. for 0.17 h. The reactions werecombined, concentrated, diluted with MTBE (1 L), and filtered. Thefilter cake was washed with MTBE (2×300 mL) and dried under reducedpressure to give the title intermediate (705 g, crude) as a yellowsolid, which was used directly in the next step. (m/z): [M+H]⁺ calcd forC₂₃H₁₈F₂N₂O₂ 393.13 found 393.1. ¹H NMR (400 MHz, DMSO-d₆) δ 14.51 (s,1H), 10.17 (d, J=3.6 Hz, 1H), 7.50 (d, J=7.2 Hz, 2H), 7.40-7.42 (m, 4H),7.24 (d, J=8.4 Hz, 1H), 7.15 (d, J=12.4 Hz, 1H), 7.06 (d, J=8.4 Hz, 1H),5.25 (s, 2H), 2.52-2.53 (m, 2H), 1.03 (t, J=7.6 Hz, 3H).

Preparation 4:5-Benzyl-2-(6-(4-(benzyloxy)-2-ethyl-5-fluorophenyl)-4-fluoro-1H-indazol-3-yl)-5H-imidazo[4,5-c]pyridine(4)

Four reactions were carried out in parallel and combined for work-up. Toa solution of6-(4-(benzyloxy)-2-ethyl-5-fluorophenyl)-4-fluoro-1H-indazole-3-carbaldehyde(3), the product of Preparation 3 (172 g, 440 mmol) in DMF (1.1 L) wasadded sodium bisulfite (68.6 g, 659 mmol) and1-benzyl-4-imino-1,4-dihydropyridin-3-amine (2) (136 g, 484 mmol) at 20°C. and the reaction mixture was stirred at 150° C. for 2 h. Fourreactions were combined and the reaction mixture was concentrated underreduced pressure. The residue was poured into water (10 L) and filtered.The filter cake was dried under reduced pressure to give the titleintermediate (990 g, crude) as a yellow solid, which was used directlywithout purification. (m/z): [M+H]⁺ calcd for C₃₅H₂₇F₂N₅O 572.2 found572.3.

Preparation 5:5-Benzyl-2-(6-(4-(benzyloxy)-2-ethyl-5-fluorophenyl)-4-fluoro-1H-indazol-3-yl)-4,5,6,7-tetrahydro-1H-imidazo[4,5-c]pyridine(5)

Three reactions were carried out in parallel and combined for work-up.To a mixture of5-benzyl-2-(6-(4-(benzyloxy)-2-ethyl-5-fluorophenyl)-4-fluoro-1H-indazol-3-yl)-5H-imidazo[4,5-c]pyridine(4), the product of Preparation 4 (330 g, 577 mmol) in methanol (1.5 L)and THF (1 L) was added sodium borohydride (267 g, 6.9 mol) in portionsat 20° C. and the reaction mixture was stirred at 20° C. for 24 h. Threereactions were combined and the reaction mixture was added to water (10L), stirred for 10 min, and filtered. The filtrate was extracted withEtOAc (2×5 L) and the combined organic phase was dried with anhydroussodium sulfate, filtered and concentrated under vacuum. The crudeproduct was diluted with EtOAc (2 L), stirred for 30 min, and filtered.The filter cake was washed with MTBE (3×200 mL) to give the titleintermediate (275 g, 28% yield) as a light yellow solid. (m/z): [M+H]⁺calcd for C₃₅H₃₁F₂N₅O 576.25 found 576.3. ¹H NMR (400 MHz, DMSO-d₆) δ7.50-7.52 (m, 2H), 7.35-7.43 (m, 7H), 7.23-7.25 (m, 3H), 7.15 (d, J=12.0Hz, 1H), 6.81 (d, J=12.0 Hz, 1H), 5.25 (s, 2H), 3.72 (s, 2H), 3.43 (br.s, 2H), 2.78 (br. s, 2H), 2.66 (br. s, 2H), 2.55 (q, 2H), 1.04 (t, J=7.6Hz, 3H).

Preparation 6:5-Ethyl-2-fluoro-4-(4-fluoro-3-(4,5,6,7-tetrahydro-1H-imidazo[4,5-c]pyridin-2-yl)-1H-indazol-6-yl)phenol(6)

Five reactions were carried out in parallel and combined for work-up. Toa mixture of5-benzyl-2-(6-(4-(benzyloxy)-2-ethyl-5-fluorophenyl)-4-fluoro-1H-indazol-3-yl)-4,5,6,7-tetrahydro-1H-imidazo[4,5-c]pyridine(5), the product of Preparation 5 (55 g, 95.5 mmol) in THF (500 mL) andmethanol (500 mL) was added palladium on carbon (15 g, 9.6 mmol) and aq.12 M HCl (10 mL). The suspension was degassed under vacuum, purged withhydrogen several times and stirred under hydrogen (50 psi) at 50° C. for12 h. The reactions were combined and the reaction mixture was filtered.The filtrate was concentrated under vacuum to provide the HCl salt ofthe title intermediate (150 g, crude) as an off-white solid. (m/z):[M+H]⁺ calcd for C₂₁H₁₉F₂N₅O 396.16 found 396.2. ¹H NMR (400 MHz, MeOD)δ 7.43 (s, 1H), 7.07 (d, J=11.6 Hz, 1H), 6.97 (d, J=11.6 Hz, 1H), 6.91(d, J=8.8 Hz, 1H), 4.57 (s, 2H), 3.74 (s, 2H), 3.24 (s, 2H), 2.55 (q,J=7.6 Hz, 2H), 1.08 (t, J=7.6 Hz, 3H).

Example 1:(1S,5R)-2-azabicyclo[3.1.0]hexan-1-yl(2-(6-(2-ethyl-5-fluoro-4-hydroxyphenyl)-4-fluoro-1H-indazol-3-yl)-6,7-dihydro-3H-imidazo[4,5-c]pyridin-5(4H)-yl)methanone(1)

To a mixture of5-ethyl-2-fluoro-4-(4-fluoro-3-(4,5,6,7-tetrahydro-3H-imidazo[4,5-c]pyridin-2-yl)-1H-indazol-6-yl)phenol(21.4 mg, 0.054 mmol) (6) and(1s,5r)-2-(tert-butoxycarbonyl)-2-azabicyclo[3.1.0]hexane-1-carboxylicacid (24.6 mg, 0.108 mmol) in DMF (0.5 ml) was added HATU (45.3 mg,0.119 mmol) and DIPEA (0.076 ml, 0.433 mmol), and the resulting solutionwas stirred at room temperature for 16 hours. To the reaction mixture,was added MeOH (2.00 ml) and water (0.500 ml), then lithium hydroxide(7.78 mg, 0.325 mmol) was added and the solution was stirred at 65° C.for 1 hour. The solution was then concentrated, and TFA (0.500 ml) wasadded to the residue. The resulting solution was stirred at roomtemperature for 30 minutes, then was concentrated. The crude product wasthen purified by preparative HPLC (5-65% acetonitrile in water gradientwith 0.1% TFA, Zorbax Bonus-RP column), to provide the TFA salt of thetitle compound (19.4 mg, 57% yield). (m/z): [M+H]⁺ calcd forC₂₇H₂₆F₂N₆O₂ 505.18 found 505.2. ¹H NMR (400 MHz, DMSO-d₆) δ 13.62 (s,1H), 12.44 (s, 1H), 9.92 (s, 1H), 7.21 (s, 1H), 7.03 (d, J=11.9 Hz, 1H),6.90 (d, J=9.1 Hz, 1H), 6.82 (d, J=11.3 Hz, 1H), 4.59 (m, 2H), 3.96 (m,1H), 3.79 (m, 1H), 3.27 (m, 1H), 2.89 (m, 1H), 2.72 (m, 2H), 2.47 (q,J=7.5 Hz, 2H), 2.06 (m, 2H), 1.92 (m, 1H), 1.30 (m, 2H), 1.00 (t, J=7.5,3H).

Preparation 7: 1-(Benzyloxy)-4-bromo-5-ethyl-2-fluorobenzene

(a) 5-Ethyl-2-fluorophenol

A mixture of compound 5-bromo-2-fluorophenol (80 g, 419 mmol) in drytetrahydrofuran (800 mL) was degassed and purged with nitrogen threetimes, and Pd(t-Bu₃P)₂ (4.28 g, 8.38 mmol) was added. Diethylzinc (114g, 921 mmol) was added to the mixture dropwise at 25° C., and thereaction mixture was stirred at 50° C. for 12 h under nitrogen andslowly poured into ice-water (1 L). EtOAc (350 mL) was added and thereaction mixture was stirred for 20 min and filtered. The filter cakewas washed with EtOAc (3×500 mL). The combined organic layers werewashed with brine (600 mL), dried over sodium sulfate, concentrated, andpurified by silica gel chromatography to give the title intermediate (85g, crude) as a yellow oil.

(b) 2-(Benzyloxy)-4-ethyl-1-fluorobenzene

To a solution of the product of the previous step (85 g, 606 mmol) inACN (850 mL) was added benzyl bromide (124 g, 728 mmol) and K₂CO₃ (126g, 909 mmol). The reaction mixture was stirred at 25° C. for 12 h,poured into water (1 L) and extracted with EtOAc (4×500 mL). Thecombined organic layers were washed with brine (600 mL), dried oversodium sulfate, concentrated, and purified by silica gel chromatographyto give the title intermediate (100 g) as a yellow oil.

(c) 1-(Benzyloxy)-4-bromo-5-ethyl-2-fluorobenzene

To a solution of the product of the previous step (100 g, 434 mmol) inACN (1.0 L) was added N-bromosuccinimide (85 g, 477 mmol) portion wise.The reaction mixture was stirred at 25° C. for 5 h, poured into water(1.3 L) and extracted with EtOAc (3×500 mL). The combined organic layerswere washed with brine (800 mL), dried over sodium sulfate,concentrated, and purified by silica gel chromatography to give thetitle compound (83 g) as yellow oil. ¹H NMR (CDCl₃, 400 MHz) δ (ppm)7.27-7.43 (m, 6H), 6.86 (d, J=8.4 Hz, 1H), 5.10 (s, 2H), 2.64 (q, J=7.6Hz, 2H), 1.15 (t, J=7.2 Hz, 1H).

Preparation 8:2-(4-(Benzyloxy)-2-ethyl-5-fluorophenyl)-4,4,5,5-tetramethyl-1,3,2-dioxaborolane

A mixture of the compound of Preparation 7 (83 g, 268 mmol),bis(pinacolato)diboron (102 g, 402 mmol), and KOAc (79.0 g, 805 mmol) indioxane (830 mL) was degassed and purged with nitrogen 3 times, andPd(dppf)Cl₂ (3.93 g, 5.37 mmol) was added. The reaction mixture wasstirred at 120° C. for 4 h under nitrogen. The mixture was cooled to 25°C., poured into water (1 L), and extracted with EtOAc (3×500 mL). Thecombined organic layers were washed with brine (800 mL), dried oversodium sulfate, and purified by silica gel chromatography. The productwas washed with methanol (200 mL), filtered, and the filter cake wasdried to give the title compound (65 g) as white solid. ¹H NMR (CDCl₃,400 MHz) δ (ppm) 7.26-7.42 (m, 5H), 6.74 (d, J=7.6 Hz, 1H), 5.08 (s,2H), 2.76 (q, J=7.2 Hz, 2H), 1.25 (s, 12H), 1.06 (t, J=7.6 Hz, 3H).

Preparation 9: 1-Benzyl-4-imino-1,4-dihydropyridin-3-amine

To a solution of pyridine-3,4-diamine (200 g, 1.8 mol) in ACN (17.0 L)was added benzyl bromide (306 g, 1.79 mol) and the reaction mixture wasstirred at 15° C. for 12 h, filtered and the filter cake was dried undervacuum to give the title compound (250 g) as a white solid. ¹H NMR(d₆-DMSO, 400 MHz) δ (ppm) 8.02 (dd, J=7.2, 1.6 Hz, 1H), 7.66 (s, 1H),7.34-7.41 (m, 5H), 6.79 (d, J=6.8 Hz, 1H), 5.62 (s, 2H), 5.36 (s, 2H).

Preparation 10:5-Benzyl-2-(6-bromo-1H-indazol-3-yl)-5H-imidazo[4,5-c]pyridine

(a) 6-Bromo-1H-indazol-3-yl-carbaldehyde

A solution of NaNO₂ (704 g, 10.2 mol) in water (1 L) was added dropwiseto a solution of 6-bromo-1H-indole (400 g, 2.0 mol) in acetone (7 L) at10° C. The reaction mixture was stirred at 10° C. for 30 min, aqueous 3MHCl (437 mL) was added slowly with vigorous stirring, keeping theinternal temperature between 10 and 25° C. The solution was stirred at20° C. for 3 h, and concentrated while keeping the temperature below 35°C. The solid was collected by filtration. The filter cake was washedwith 1:2 petroleum ether:MTBE (800 mL). The solids were collected byfiltration and dried under vacuum to afford the title intermediate (450g) as a black brown solid. ¹H NMR (CH₃OD, 400 MHz) δ (ppm) 7.77 (d,J=8.8 Hz, 1H), 7.69 (s, 1H), 7.22 (dd, J=8.4, 2.4 Hz, 1H), 5.70 (s, 1H).

(b) 5-Benzyl-2-(6-bromo-1H-indazol-3-yl)-5H-imidazo[4,5-c]pyridine

To a stirred solution of 6-bromo-1H-indazol-3-yl-carbaldehyde (150.0 g,666 mmol) and 1-benzyl-4-imino-1,4-dihydropyridin-3-amine (127.5 g,639.9 mmol) in DMF (750 mL) was charged NaHSO₃ (83.2 g, 799.9 mmol) andthe reaction mixture was stirred for 6 h at 140° C. and poured intowater (3.5 L). The precipitate was filtered and washed with water (1 L)to give the title compound (180 g) as a black brown solid. ¹H NMR(d₆-DMSO, 400 MHz) δ (ppm) 8.69 (s, 1H) 8.71 (d, J=7.2 Hz, 1H) 8.37 (d,J=8.4 Hz, 1H) 8.07 (d, J=6.4 Hz, 1H) 7.97 (s, 1H) 7.38-7.43 (m, 3H)7.50-7.54 (m, 4H) 5.87 (s, 2H).

Preparation 11:5-Benzyl-2-(6-bromo-1H-indazol-3-yl)-4,5,6,7-tetrahydro-1H-imidazo[4,5-c]pyridine

To a solution of5-benzyl-2-(6-bromo-1H-indazol-3-yl)-5H-imidazo[4,5-c]pyridine (23.0 g,56.9 mmol) in MeOH (200 mL) and THF (1 L) was added NaBH₄ (12.9 g, 341.3mmol) portion-wise and the reaction mixture was stirred at 50° C. for 2h. Acetic acid (10 eq) was added, the solution was concentrated todryness and purified by silica gel chromatography (30 g silica, 0-10%MeOH/DCM with 0.1% TEA) to give the title compound (6.0 g). ¹H NMR(d₆-DMSO, 400 MHz) δ (ppm) 8.24 (d, J=8.0 Hz, 1H), 7.77 (s, 1H),7.28-7.37 (m, 7H), 3.74 (s, 2H), 3.48 (br.s, 2H), 2.80 (s, 2H), 2.66 (s,2H).

Preparation 12:5-Benzyl-2-(6-(4-(benzyloxy)-2-ethyl-5-fluorophenyl)-1H-indazol-3-yl)-4,5,6,7-tetrahydro-1H-imidazo[4,5-c]pyridine

(a) tert-Butyl5-benzyl-2-(6-bromo-1-(tert-butoxycarbonyl)-1H-indazol-3-yl)-4,5,6,7-tetrahydro-1H-imidazo[4,5-c]pyridine-1-carboxylate

Two reactions were carried out in parallel. A suspension of5-benzyl-2-(6-bromo-1H-indazol-3-yl)-4,5,6,7-tetrahydro-1H-imidazo[4,5-c]pyridine(80 g, 196 mmol), di-tert-butyl dicarbonate (128 g, 587.8 mmol, 135 mL)and TEA (79.3 g, 784 mmol, 109 mL) in DCM (1 L) was stirred at 20° C.for 12 h. The two reaction suspensions were combined, concentrated todryness, and purified by silica gel chromatography (petroleumether:EtOAc 10:1-0:1) to give the title intermediate (170.0 g).

(b)5-Benzyl-2-(6-bromo-1H-indazol-3-yl)-4,5,6,7-tetrahydro-1H-imidazo[4,5-c]pyridine

Two reactions were carried out in parallel. A solution of the product ofthe previous step (85 g, 140 mmol) and 4M HCl in MeOH (400 mL) in DCM(400 mL) was stirred at 25° C. for 12 h. The reaction mixtures werecombined and concentrated to dryness, DCM (250 mL) was added withstirring, and the reaction mixture was stirred for 30 min and filtered.The filter cake was washed with DCM (2×20 mL) and dried to give thetitle compound (85 g) as an off-white solid.

(c)5-Benzyl-2-(6-(4-(benzyloxy)-2-ethyl-5-fluorophenyl)-1H-indazol-3-yl)-4,5,6,7-tetrahydro-1H-imidazo[4,5-c]pyridine

Eighty-five reactions were carried out in parallel. The product of theprevious step (1.0 g, 2.5 mmol),2-(4-(benzyloxy)-2-ethyl-5-fluorophenyl)-4,4,5,5-tetramethyl-1,3,2-dioxaborolane(873 mg, 2.5 mmol), and Pd(PPh₃)₄ (227 mg, 196 μmol) were dissolved in amixture of water (4 mL) and dioxane (10 mL). The reaction vial wasbubbled with nitrogen for 2 min and Na₂CO₃ (779 mg, 7.4 mmol) was addedquickly under nitrogen. The reaction mixture was heated at 130° C. for1.5 h. The 85 reaction mixtures were combined and concentrated underreduced pressure. The residue was dissolved in DCM (500 mL) and purifiedby silica gel chromatography (150 g silica, eluted with DCM:THF (6:1 to3:1)) to give compound the title compound (50 g) as an off-white solid.

Preparation 13:5-Ethyl-2-fluoro-4-(3-(4,5,6,7-tetrahydro-1H-imidazo[4,5-c]pyridin-2-yl)-1H-indazol-6-yl)phenol

A mixture of5-benzyl-2-(6-(4-(benzyloxy)-2-ethyl-5-fluorophenyl)-1H-indazol-3-yl)-4,5,6,7-tetrahydro-1H-imidazo[4,5-c]pyridine(44.5 g, 79.8 mmol), Pd(OH)₂/C (25 g, 2.7 mmol, 50% purity) and TFA(44.5 g, 390 mmol, 28.9 mL) in MeOH (500 mL) was stirred under hydrogen(50 Psi) for 4 h and filtered. Pd(OH)₂/C (25 g, 2.7 mmol, 50% purity)was added to the filtrate and the resulting suspension was stirred underhydrogen (50 Psi) at 25° C. for 12 h. The suspension was combined withthe suspension from a prior reaction at the 5.5 g scale and filtered.The filter cake was washed with 20:1 MeOH:TFA (2×200 mL). The combinedfiltrate was concentrated and 4 M HCl in MeOH (200 mL) was added to theresidue with stirring. The resulting suspension was concentrated,slurried with MeOH (80 mL) and stirred for 30 min. A white solidprecipitated. The solid was filtered, the filter cake was washed withMeOH (2×10 mL) and dried under vacuum to give the HCl salt of the titlecompound (24.8 g) as an off-white solid. (m/z): [M+H]⁺ calcd forC₂₁H₂₀FN₅O 378.17 found 378.1. ¹H NMR (d₆-DMSO, 400 MHz) δ (ppm) 8.23(d, J=8.4 Hz, 1H), 7.59 (s, 1H), 7.35 (d, J=11.2 Hz, 1H), 6.90-6.97 (m,2H), 4.57 (s, 2H), 3.72 (t, J=6.0 Hz, 2H), 3.22 (t, J=6.0 Hz, 2H), 2.51(q, J=7.6 Hz, 2H), 1.04 (t, J=7.6 Hz, 3H).

Example 2:((1S,5R)-2-azabicyclo[3.1.0]hexan-1-yl)(2-(6-(2-ethyl-5-fluoro-4-hydroxyphenyl)-1H-indazol-3-yl)-1,4,6,7-tetrahydro-5H-imidazo[4,5-c]pyridin-5-yl)methanoneC-1

5-ethyl-2-fluoro-4-(3-(4,5,6,7-tetrahydro-1H-imidazo[4,5-c]pyridin-2-yl)-1H-indazol-6-yl)phenol(4.0 g, 10.60 mmol),(1s,5r)-2-(tert-butoxycarbonyl)-2-azabicyclo[3.1.0]hexane-1-carboxylicacid (3.61 g, 15.90 mmol), and DIPEA (7.40 ml, 42.4 mmol) were dissolvedin DMF (60 ml), then HATU (8.06 g, 21.20 mmol) was added and thereaction mixture was stirred at room temperature for 24 hours. Hydrazine(0.665 ml, 21.20 mmol) was then added to cleave undesired byproducts,then the reaction mixture was concentrated to about 20 mL. This solutionwas then dripped into 200 mL of water to precipitate out the product,which was then collected by filtration and dried under vacuum. Theresulting solid was dissolved in dioxane (40 ml) and water (8 ml), thenhydrochloric acid, 4M in dioxane (40 ml, 160 mmol) was added and thereaction mixture was stirred at room temperature for 2 hours. Thesolution was then frozen and lyophilized, and the resulting solid waspurified by preparative HPLC (5-70% acetonitrile/water gradient, C18column) to provide the TFA salt of the title compound (2.69 g, 42%yield). (m/z): [M+H]⁺ calcd for C₂₇H₂₇FN₆O₂ 487.55 found 487.7. ¹H NMR(400 MHz, DMSO-d₆) δ 13.15 (s, 1H), 12.42 (s, 1H), 9.79 (s, 1H), 8.29(d, J=8.4 Hz, 1H), 7.34 (s, 1H), 7.07 (d, J=8.4 Hz, 1H), 6.99 (d, J=11.9Hz, 1H), 6.88 (d, J=9.1 Hz, 1H), 4.51 (m, 2H), 3.92 (m, 2H), 3.99 (m,1H), 2.65 (m, 3H), 2.47 (q, J=7.5 Hz, 2H), 1.89 (m, 1H), 1.72 (m, 2H),0.98 (t, J=7.5 Hz, 3H), 0.89 (m, 2H).

Biological Assays

Compound 1 has been characterized in one or more of the followingbiological assays.

Assay 1: Biochemical JAK Kinase Assays

A panel of four LanthaScreen JAK biochemical assays (JAK1, 2, 3 andTyk2) were carried in a common kinase reaction buffer (50 mM HEPES, pH7.5, 0.01% Brij-35, 10 mM MgCl₂, and 1 mM EGTA). Recombinant GST-taggedJAK enzymes and a GFP-tagged STAT1 peptide substrate were obtained fromLife Technologies.

Serially diluted compound was pre-incubated with each of the four JAKenzymes and the substrate in white 384-well microplates (Corning) atambient temperature for 1 h. ATP was subsequently added to initiate thekinase reactions in 10 μL total volume, with 1% DMSO. The final enzymeconcentrations for JAK1, 2, 3 and Tyk2 are 4.2 nM, 0.1 nM, 1 nM, and0.25 nM respectively; the corresponding Km ATP concentrations used are25 μM, 3 μM, 1.6 μM, and 10 μM; while the substrate concentration is 200nM for all four assays. Kinase reactions were allowed to proceed for 1hour at ambient temperature before a 10 μL preparation of EDTA (10 mMfinal concentration) and Tb-anti-pSTAT1 (pTyr701) antibody (LifeTechnologies, 2 nM final concentration) in TR-FRET dilution buffer (LifeTechnologies) was added. The plates were allowed to incubate at ambienttemperature for 1 h before being read on the EnVision reader (PerkinElmer). Emission ratio signals (520 nm/495 nm) were recorded andutilized to calculate the percent inhibition values based on DMSO andbackground controls.

For dose-response analysis, percent inhibition data were plotted vs.compound concentrations, and IC₅₀ values were determined from a4-parameter robust fit model with the Prism software (GraphPadSoftware). Results were expressed as pIC₅₀ (negative logarithm of IC₅₀)and subsequently converted to pK_(i) (negative logarithm of dissociationconstant, Ki) using the Cheng-Prusoff equation.

Compound 1 exhibited the following enzyme potency.

TABLE 1 JAK 1 pK_(i) JAK 2 pK_(i) JAK 3 pK_(i) Tyk 2 pK_(i) 9.9 10.2 9.68.6Assay 2: Inhibition of IL-2 Stimulated pSTAT5 in Tall-1 T Cells

The potency of test compounds for inhibition of interleukin-2 (IL-2)stimulated STAT5 phosphorylation was measured in the Tall-1 human T cellline (DSMZ) using AlphaLisa. Because IL-2 signals through JAK1/3, thisassay provides a measure of JAK1/3 cellular potency.

Phosphorylated STAT5 was measured via the AlphaLISA SureFire UltrapSTAT5 (Tyr694/699) kit (PerkinElmer). Human T cells from the Tall-1cell line were cultured in a 37° C., 5% CO₂ humidified incubator in RPMI(Life Technologies) supplemented with 15% Heat Inactivated Fetal BovineSerum (FBS, Life Technologies), 2 mM Glutamax (Life Technologies), 25 mMHEPES (Life Technologies) and 1× Pen/Strep (Life Technologies).Compounds were serially diluted in DMSO and dispensed acoustically toempty wells. Assay media (phenol red-free DMEM (Life Technologies)supplemented with 10% FBS (ATCC)) was dispensed (4 μL/well) and platesshaken at 900 rpm for 10 mins. Cells were seeded at 45,000 cells/well inassay media (4 μL/well), and incubated at 37° C., 5% CO₂ for 1 hour,followed by the addition of IL-2 (R&D Systems; final concentration 300ng/mL) in pre-warmed assay media (4 μL) for 30 minutes. After cytokinestimulation, cells were lysed with 6 ul of 3× AlphaLisa Lysis Buffer(PerkinElmer) containing 1× PhosStop and Complete tablets (Roche). Thelysate was shaken at 900 rpm for 10 minutes at room temperature (RT).Phosphorylated STAT5 was measured via the pSTAT5 AlphaLisa kit(PerkinElmer). Freshly prepared acceptor bead mixture was dispensed ontolysate (5 μL) under green filtered <100 lux light. Plates were shaken at900 rpm for 2 mins, briefly spun down, and incubated for 2 hrs at RT inthe dark. Donor beads were dispensed (5 μL) under green filtered <100lux light. Plates were shaken at 900 rpm for 2 minutes, briefly spundown, and incubated overnight at RT in the dark. Luminescence wasmeasured with excitation at 689 nm and emission at 570 nm using anEnVision plate r reader (PerkinElmer) under green filtered <100 luxlight.

To determine the inhibitory potency of test compounds in response toIL-2, the average emission intensity of beads bound to pSTAT5 wasmeasured in a human T cell line. IC₅₀ values were determined fromanalysis of the inhibition curves of signal intensity versus compoundconcentration. Data are expressed as pIC₅₀ (negative decadic logarithmIC₅₀) values (mean±standard deviation). Compound 1 exhibited a pIC₅₀value of 7.8 in this assay.

Assay 3: Cellular JAK Potency Assay: Inhibition of IL-13 StimulatedpSTAT6 in BEAS-2B Cells

The AlphaScreen JAKI cellular potency assay was carried out by measuringinterleukin-13 (IL-13, R&D Systems) induced STAT6 phosphorylation inBEAS-2B human lung epithelial cells (ATCC). The anti-STAT6 antibody(Cell Signaling Technologies) was conjugated to AlphaScreen acceptorbeads (Perkin Elmer), while the anti-pSTAT6 (pTyr641) antibody (CellSignaling Technologies) was biotinylated using EZ-Link Sulfo-NHS-Biotin(Thermo Scientific).

BEAS-2B cells were grown at 37° C. in a 5% CO₂ humidified incubator in50% DMEM/50% F-12 medium (Life Technologies) supplemented with 10% FBS(Hyclone), 100 U/mL penicillin, 100 μg/mL streptomycin (LifeTechnologies), and 2 mM GlutaMAX (Life Technologies). On day 1 of theassay, cells were seeded at a 7,500 cells/well density in whitepoly-D-lysine-coated 384-well plates (Corning) with 25 μL medium, andwere allowed to adhere overnight in the incubator. On day 2 of theassay, the medium was removed and replaced with 12 μL of assay buffer(Hank's Balanced Salt Solution/HBSS, 25 mM HEPES, and 1 mg/mL bovineserum albumin/BSA) containing dose-responses of test compounds. Thecompound was serially diluted in DMSO and then diluted another 1000-foldin media to bring the final DMSO concentration to 0.1%. Cells wereincubated with test compounds at 37° C. for 1 h, and followed by theaddition of 12 μl of pre-warmed IL-13 (80 ng/mL in assay buffer) forstimulation. After incubating at 37° C. for 30 min, the assay buffer(containing compound and IL-13) was removed, and 10 μL of cell lysisbuffer (25 mM HEPES, 0.1% SDS, 1% NP-40, 5 mM MgCl₂, 1.3 mM EDTA, 1 mMEGTA, and supplement with Complete Ultra mini protease inhibitors andPhosSTOP from Roche Diagnostics). The plates were shaken at ambienttemperature for 30 min before the addition of detection reagents. Amixture of biotin-anti-pSTAT6 and anti-STAT6 conjugated acceptor beadswas added first and incubated at ambient temperature for 2 h, followedby the addition of streptavidin conjugated donor beads (Perkin Elmer).After a minimum of 2 h incubation, the assay plates were read on theEnVision plate reader. AlphaScreen luminescence signals were recordedand utilized to calculate the percent inhibition values based on DMSOand background controls.

For dose-response analysis, percent inhibition data were plotted vs.compound concentrations, and IC₅₀ values were determined from a4-parameter robust fit model with the Prism software. Results may alsobe expressed as the negative logarithm of the IC₅₀ value, pIC₅₀.Compound 1 exhibited a pIC₅₀ value of 8 in this assay.

Assay 4: Cellular JAK Potency Assay: Inhibition of IL-2/Anti-CD3Stimulated IFNγ in Human PBMCs

The potency of the test compound for inhibition of interleukin-2(IL-2)/anti-CD3 stimulated interferon gamma (IFNγ) was measured in humanperipheral blood mononuclear cells (PBMCs) isolated from human wholeblood (Stanford Blood Center). Because IL-2 signals through JAK, thisassay provides a measure of JAK cellular potency.

(1) Human peripheral blood mononuclear cells (PBMC) were isolated fromhuman whole blood of healthy donors using a ficoll gradient. Cells werecultured in a 37° C., 5% CO₂ humidified incubator in RPMI (LifeTechnologies) supplemented with 10% Heat Inactivated Fetal Bovine Serum(FBS, Life Technologies), 2 mM Glutamax (Life Technologies), 25 mM HEPES(Life Technologies) and 1× Pen/Strep (Life Technologies). Cells wereseeded at 200,000 cells/well in media (50 μL) and cultured for 1 h. Thecompound was serially diluted in DMSO and then diluted another 500-fold(to a 2× final assay concentration) in media. The test compounddilutions (100 μL/well) were added to cells, and incubated at 37° C., 5%CO₂ for 1 h, followed by the addition of IL-2 (R&D Systems; finalconcentration 100 ng/mL) and anti-CD3 (BD Biosciences; finalconcentration 1 μg/mL) in pre-warmed assay media (50 μL) for 24 h.

(2) After cytokine stimulation, cells were centrifuged at 500 g for 5min and supernatants removed and frozen at −80° C. To determine theinhibitory potency of test compounds in response to IL-2/anti-CD3,supernatant IFNγ concentrations were measured via ELISA (R&D Systems).IC₅₀ values were determined from analysis of the inhibition curves ofconcentration of IFNγ vs compound concentration. Data are expressed aspIC₅₀ (negative decadic logarithm IC₅₀) values. Compound 1 exhibited apIC₅₀ value of 6.7 in this assay.

Assay 5: Cellular JAK Potency Assay: Inhibition of IL-2 StimulatedpSTAT5 in CD4+ T Cells

The potency of the test compound for inhibition of interleukin-2(IL-2)/anti-CD3 stimulated STAT5 phosphorylation was measured inCD4-positive (CD4+) T cells in human peripheral blood mononuclear cells(PBMCs) isolated from human whole blood (Stanford Blood Center) usingflow cytometry. Because IL-2 signals through JAK, this assay provides ameasure of JAK cellular potency.

CD4+ T cells were identified using a phycoerythrobilin (PE) conjugatedanti-CD4 antibody (Clone RPA-T4, BD Biosciences), while an Alexa Fluor647 conjugated anti-pSTAT5 antibody (pY694, Clone 47, BD Biosciences)was used to detect STAT5 phosphorylation.

(1) The protocol of Assay 4 paragraph (1) was followed with theexception that the cytokine stimulation with IL-2/anti-CD3 was performedfor 30 min instead of 24 h.

(2) After cytokine stimulation, cells were fixed with pre warmed fixsolution (200 μL; BD Biosciences) for 10 min at 37° C., 5% CO₂, washedtwice with DPBS buffer (1 mL, Life Technologies), and resuspended in icecold Perm Buffer III (1000 μL, BD Biosciences) for 30 min at 4° C. Cellswere washed twice with 2% FBS in DPBS (FACS buffer), and thenresuspended in FACS buffer (100 μL) containing anti-CD4 PE (1:50dilution) and anti-CD3 anti-CD3Alexa Fluor 647 (1:5 dilution) for 60 minat room temperature in the dark. After incubation, cells were washedtwice in FACS buffer before being analyzed using a LSRII flow cytometer(BD Biosciences). To determine the inhibitory potency of the testcompound in response to IL-2/anti-CD3, the median fluorescent intensity(MFI) of pSTAT5 was measured in CD4+ T cells. IC₅₀ values weredetermined from analysis of the inhibition curves of MFI vs compoundconcentration.

Data are expressed as pIC₅₀ (negative decadic logarithm IC₅₀) values.Compound 1 exhibited a pIC₅₀ value of 7.7 in this assay.

Assay 6: Cellular JAK Potency Assay: Inhibition of IL-6 Stimulated CCL2(MCP-1) in Human PBMCs

The potency of the test compound for inhibition of interleukin-6 (IL-6)stimulated CCL2 (MCP-1) production was measured in human peripheralblood mononuclear cells (PBMCs) isolated from human whole blood(Stanford Blood Center). Because IL-6 signals through JAK, this assayprovides a distal measure of JAK cellular potency.

(1) The protocol of Assay 4 paragraph (1) was followed up to theincubation with test compounds. In the present assay, after testcompounds were added to wells and incubated, IL-6 (R&D Systems; finalconcentration 10 ng/ml) in pre-warmed assay media (50 μL) was added.

(2) After cytokine stimulation for 48 h, cells were centrifuged at 500 gfor 5 min and supernatants were removed and frozen at −80° C. Todetermine the inhibitory potency of the test compound in response toIL-6, supernatant CCL2 (MCP-1) concentrations were measured via ELISA(R&D Systems). IC₅₀ values were determined from analysis of theinhibition curves of concentration of CCL2/MCP-1 vs compoundconcentration. Data are expressed as pIC₅₀ (negative decadic logarithmIC₅₀) values. Compound 1 exhibited a pIC₅₀ value of 6.5 in this assay.

Assay 7: Pharmacodynamic Assay: Inhibition of IL6-Induced pSTAT3 in Rats

The ability of a single intravitreal administration of test compound toinhibit IL-6 induced pSTAT3 was measured in rat retina/choroidhomogenates.

A solution formulation was prepared by dissolving compound 1 in 1% HPMCE5+15% HPβCD, pH7 to attain a target concentration of 10 mg/mL. FemaleLewis rats were intravitreally (IVT) dosed (5 μL per eye) with eitherthe vehicle, vehicle plus rat IL-6 (Peprotech; 0.5 μg), or a combinationof IL-6 plus compound (0.5 μg IL-6 and 45 μg compound 1). Ocular tissueswere dissected one hour after the IVT injection. The retina/choroidtissues were homogenized and pSTAT3 levels were measured using anAlphaLisa (PerkinElmer). The percent inhibition of IL-6-induced pSTAT3was calculated in comparison to the vehicle and IL-6 groups.

A 45 μg dose of compound 1 inhibited IL-6-induced pSTAT3 by 84% in theretina/choroid homogenates.

Assay 8: Lung and Plasma Pharmacokinetics in Mice

Plasma and lung concentrations of compound 1 and ratios thereof weredetermined in the following manner. BALB/c mice from Charles RiverLaboratories were used in the assay. Compound 1 was formulated in 20%propylene glycol in citrate buffer (pH 4) at a concentration of 0.324mg/mL as a solution. 50 μL of the solution formulation was introducedinto the trachea of a mouse by oral aspiration. At various time points(0.167, 2, 6 hr) post dosing, blood samples were removed via cardiacpuncture and intact lungs were excised from the mice. Blood samples werecentrifuged (Eppendorf centrifuge, 5804R) for 4 minutes at approximately12,000 rpm at 4° C. to collect plasma. Lungs were padded dry, weighed,and homogenized in 0.6 mL sterile water. Plasma and lung concentrationsof compound 1 were determined by LC-MS analysis against analyticalstandards constructed into a standard curve in the test matrix.Sustained exposure in lungs was found with a lung AUC (0-6 hr) of 43.5μg hr/g. The lung-to-plasma ratio was determined as the ratio of thelung AUC in μg hr/g to the plasma AUC in μg hr/mL (where AUC isconventionally defined as the area under the curve of test compoundconcentration vs. time). The lung-to-plasma AUC ratio was 59.6,highlighting the high lung exposure versus plasma.

Assay 9: Kinome Screen and GINI Coefficient

Compounds 1 and C-1 were screened against other kinases to evaluatetheir selectivity profile.

Kinase-tagged T7 phage strains were grown in parallel in 24-well blocksin an E. coli host derived from the BL21 strain. E. coli were grown tolog-phase and infected with T7 phage from a frozen stock (multiplicityof infection=0.4) and incubated with shaking at 32° C. until lysis(90-150 minutes). The lysates were centrifuged (6,000×g) and filtered(0.2 μm) to remove cell debris. The remaining kinases were produced inHEK-293 cells and subsequently tagged with DNA for qPCR detection.

Streptavidin-coated magnetic beads were treated with biotinylated smallmolecule ligands for 30 minutes at room temperature to generate affinityresins for kinase assays. The liganded beads were blocked with excessbiotin and washed with blocking buffer (SeaBlock (Pierce), 1% BSA, 0.05%Tween 20, 1 mM DTT) to remove unbound ligand and to reduce non-specificphage binding. Binding reactions were assembled by combining kinases,liganded affinity beads, and test compounds in 1× binding buffer (20%SeaBlock, 0.17×PBS, 0.05% Tween 20, 6 mM DTT). Test compounds wereprepared as 40× stocks in 100% DMSO and directly diluted into the assay.All reactions were performed in polypropylene 384-well plates in a finalvolume of 0.04 ml. The assay plates were incubated at room temperaturewith shaking for 1 hour and the affinity beads were washed with washbuffer (1×PBS, 0.05% Tween 20). The beads were then re-suspended inelution buffer (1×PBS, 0.05% Tween 20, 0.5 μM non-biotinylated affinityligand) and incubated at room temperature with shaking for 30 minutes.The kinase concentration in the eluates was measured by qPCR.

The compounds were screened at 1 μM, and results for primary screenbinding interactions in Table 2 and 3 are reported as “% inhibition”(=100−((test compound signal−positive control signal)/((negative controlsignal)−(positive control signal))×100) where the negative control isDMSO and the positive control is a control compound.

TABLE 2 Kinase Compound ALK AURKA CDK2 CDK7 CDK9 CSF1R EPHB6 GSK3B 1 7926 1 82 13 98 85 32 C-1 87 54 50 98.8 89 100 96.5 52

TABLE 3 Kinase PKAC- Compound KIT PAK4 ALPHA PLK4 SLK SRC SYK VEGFR2CSNK1D 1 83 41 22 39 100 65 44 47 25 C-1 98.5 89 55 40 99.9 98.9 82 5966

Compound 1 was found to exhibit significantly lower binding inhibitionfor EPHB6, KIT, PAK4, SRC, CDK7 and CDK9 than compound C-1. Compound 1also had lower binding inhibition for several other kinases.

Both compounds 1 and C-1 were screened against 35 different kinases. TheGini coefficient was determined for both compounds. Compound 1 had aGINI coefficient of 0.60 and compound C-1 had a GINI coefficient of0.46. The Gini coefficient is used to express the selectivity of acompound against a panel of kinases (Graczyk, J. Med. Chem., 2007, 50,5773-5779). A higher number corresponds to a more selective compound.

The only structural difference between compound 1 and compound C-1 isthe presence of a fluoro group on the core. This structural differencehas been shown to have an important effect on the kinome selectivity ofthe compound.

Assay 10: Cell Viability Assay

A CellTiter-Glo luminescent cell viability/cytotoxicity assay wascarried out in BEAS-2B human lung epithelial cells (ATCC) under thenormal growth condition.

Cells were grown at 37° C. in a 5% CO₂ humidified incubator in 50%DMEM/50% F-12 medium (Life Technologies) supplemented with 10% FBS(Hyclone), 100 U/mL penicillin, 100 μg/mL streptomycin (LifeTechnologies), and 2 mM GlutaMAX (Life Technologies). On day 1 of theassay, cells were seeded at a 500 cells/well density in white 384-welltissue culture plates (Corning) with 25 μL medium, and were allowed toadhere overnight in the incubator. On day 2 of the assay, 5 μL of mediumcontaining dose-responses of test compounds was added, and incubated at37° C. for 48 h. 30 μL of CellTiter-Glo detection solution (Promega) wassubsequently added, mixed on an orbital shaker for 5 min, and incubatedfor additional 10 min before being read on the EnVision reader.Luminescence signals were recorded and percent DMSO control values werecalculated.

For dose-response analysis, percent DMSO control data were plotted vs.compound concentrations to derive dose-response curves by lineconnecting each data point. The concentration at which each curvecrosses the 15% inhibition threshold is defined as CC₁₅.

It is expected that test compounds exhibiting a higher CC₁₅ value inthis assay have less likelihood to cause cytotoxicity.

Compound 1 exhibited a pCC₁₅ of <5 (corresponding to a CC₁₅ of >10 μMwhereas compound C-1 exhibited a pCC₁₅ of 5.8 (corresponding to a CC₁₅of 1.58 μM). Therefore, compound 1 is significantly less likely to causecytotoxicity than compound C-1 based on this assay.

The only structural difference between compound 1 and compound C-1 isthe presence of a fluoro group on the core. This structural differencehas been shown to have an important effect on the cytotoxicity of thecompound.

Assay 10: Cellular JAK Potency Assay: Inhibition of IL-6 Trans-SignalingStimulated p-STAT3 in Primary Human Retinal Microvascular EndothelialCells

The cellular potency of the test compound toward inhibition of IL-6trans-signaling (IL6+sIL6Rα)-induced STAT3 phosphorylation was measuredin primary human retinal microvascular endothelial cells (HRMECs). IL-6trans-signaling is mediated by IL-6 receptor beta (gp130), which isintracellularly coupled to JAK enzymes. Following gp130 engagement anddimerization, JAKs are activated and directly phosphorylate STAT3. Thus,this assay provides a measure of JAK cellular potency. HRMECs (CellSystems, Kirkland Wash.) were plated in sterile, tissue culture treated,flat-bottom 96-well assay plates coated with Attachment Factor™ (CellSystems, Kirkland Wash.) at a density of 1×10⁴ cells per well. HRMECcultures were grown for 3 days at 37° C., 95% humidity, 5% CO₂ inculture media supplemented with 10% fetal bovine serum, Culture Boost™growth factors, and BacOff® antibiotic (Cell Systems, Kirkland Wash.).The test compound was serially diluted in DMSO and then prepared at1.11× final assay concentration in complete culture media. Cells wereincubated with 90 μL of 1.11× test compound for 1 hour at 37° C.followed by the addition of 10 μL 10×IL-6/sIL6Rα (PeproTech, Inc.; finalconcentration 500 pM IL-6, 5 nM sIL6Rα) in pre-warmed assay media for 30minutes. Following stimulation, supernatatants were removed and thecellular material was collected in 50 μL lysis buffer (AlphaLISA®SureFire® UltraT^(M) assay kit, PerkinElmer) containing phosphataseinhibtors (PhosSTOP™, Roche) and stored at −80° C. until analysis. Todetermine the inhibitory potency of the test compound, levels of p-STAT3were measured using AlphaLISA® SureFire® Ultra™ p-STAT3 (Tyr705) assaykit (PerkinElmer) according to manufacturer's instructions. IC₅₀ valueswere determined from inhibition curves of % p-STAT3 Alpha Signal vs testcompound concentration (curve fitting conducted with GraphPad Prism7.0). Data are expressed as pIC₅₀ (negative log₁₀(test compoundconcentration)). Compound 1 exhibited a pIC₅₀ of 6.3±0.1 in threeindependent experiments.

While the present invention has been described with reference tospecific aspects or embodiments thereof, it will be understood by thoseof ordinary skilled in the art that various changes can be made orequivalents can be substituted without departing from the true spiritand scope of the invention. Additionally, to the extent permitted byapplicable patent statutes and regulations, all publications, patentsand patent applications cited herein are hereby incorporated byreference in their entirety to the same extent as if each document hadbeen individually incorporated by reference herein.

1. A compound of formula:

or a pharmaceutically-acceptable salt thereof.
 2. A compound of formula:


3. A pharmaceutical composition comprising the compound of claim 1, anda pharmaceutically-acceptable carrier.
 4. The pharmaceutical compositionof claim 3, wherein the composition is suitable for application to theeye.
 5. The pharmaceutical composition of claim 4, wherein thecomposition is suitable for intravitreal injection.
 6. Thepharmaceutical composition of claim 5, wherein the composition is asuspension.
 7. A process for preparing a compound of formula 1

or a pharmaceutically-acceptable salt thereof, the process comprising:(a) reacting a compound of formula 6:

with a compound of formula 7-PG:

wherein R^(A) is hydroxyl or a leaving group and PG is anamino-protecting group, to give compound 1-PG:

(b) deprotecting compound 1-PG; and (c) optionally preparing apharmaceutically-acceptable salt to provide a compound of formula 1, ora pharmaceutically-acceptable salt thereof.
 8. The process of claim 7,wherein R^(A) is hydroxyl.
 9. The process of claim 8, wherein thereaction between 6 and 7-PG is conducted in the presence of HATU. 10.The process of claim 7, wherein PG is Boc. 11-16. (canceled)
 17. Amethod of treating an ocular disease in a mammal, the method comprisingadministering a pharmaceutical composition comprising the compound ofclaim 1, and a pharmaceutically-acceptable carrier, to the eye of themammal.
 18. The method of claim 17, wherein the ocular disease isselected from the group consisting of uveitis, diabetic retinopathy,diabetic macular edema, dry eye disease, age-related maculardegeneration, retinal vein occlusion, and atopic keratoconjunctivitis.19. The method of claim 18 wherein the ocular disease is diabeticmacular edema.
 20. The method of claim 18 wherein the ocular disease isuveitis.
 21. The method of claim 18 wherein the ocular disease isdiabetic retinopathy.
 22. The method of claim 18 wherein the oculardisease is dry eye disease.
 23. The method of claim 18 wherein theocular disease is age-related macular degeneration
 24. The method ofclaim 18 wherein the ocular disease is retinal vein occlusion
 25. Themethod of claim 18 wherein the ocular disease is atopickeratoconjunctivitis.